Compositions and methods of use for antibodies of dickkopf-1

ABSTRACT

Antibodies and fragments that bind to the protein target Dickkopf (DKK1) are provided, as are methods of use and kits, for treating a target cell, in particular, a cell associated with an osteolytic condition.

PRIORITY INFORMATION

This U.S. application is a divisional application of Ser. No.14/163,230, filed Jan. 24, 2014, which is a divisional application ofSer. No. 12/160,875, filed Jan. 12, 2007, which claims priority to U.S.Provisional Application No. 60/759,216, filed Jan. 13, 2006, thecontents of which are incorporated herein by reference in theirentirety.

FIELD OF USE

The present invention relates to compositions and methods of use forantibodies of Dickkopf-1 (“DKK1”), Dickkopf-4 (“DKK4”), or both, totreat DKK-related abnormalities of bone density, metabolism, diabetes,cancers and the like.

BACKGROUND OF THE INVENTION

The Wnt signaling pathway are involved in the control of embryonicdevelopment and neoplastic processes. Extracellular Wnt proteins areresponsible for the growth and differentiation of many cell types,including in particular the growth, differentiation and regulation ofosteoblasts, osteoclasts and adipocytes.

Many cancers are associated with bone tissues and can result inosteoblastic lesions such as those found in prostate cancer, or inosteolytic lesions such as those found in lung cancer, breast cancer,and multiple myeloma (e.g., Tian et al., 2003 New England J. Med.349:2483-2494).

Members of the Wnt gene family encode secreted glycoproteins that arerequired for a variety of developmental processes (Fedi et al., 1999 J.Bio. Chem. 274:19465-19472). A Wnt family member protein initiates asignaling pathway that is important for the growth and differentiationof osteoblasts, which cause bone deposition. In addition to boneformation, bone resorption is an ongoing normal process conducted bycells known as osteoclasts. In contrast, receptor activator of nuclearfactor-kappa B ligand (RANKL) is the final mediator of osteoclastic boneresorption, where it plays a major role in the pathogenesis ofpostmenopausal osteoporosis, as well as in bone loss associated withrheumatoid arthritis, metastatic cancer, multiple myeloma, aromataseinhibitor therapy and androgen deprivation therapy (see, e.g., Lewiecki(2006) Expert Opin Biol Ther. 6: 1041-50). Osteoprotegrin (OPG), whichis expressed by osteoblasts, inhibits RANKL, thereby decreasingosteoclast activity and formation. The balance between anabolic boneformation and analytic bone resorption regulates normal bone density,while an increase in one or the other leads to increased bone density orincreased bone loss, respectively.

Wnt binds and acts through other cell surface proteins and Wnt signalingcan contribute to the neoplastic process. Furthermore, geneticalterations can affect a cellular protein complex known as adenomatouspolyposis coli involving a protein β-catenin, and these complexes havebeen observed in cells of patients with diseases such as human coloncancer, melanomas, and hepatocellular carcinomas, indicating thataberrations of Wnt signaling pathways are relevant to the development ofthese and possibly other human cancers (Fedi et al., 1999 J. Bio. Chem.274:19465-19472).

There are at least two families of proteins that inhibit Wnt signaling,namely the secreted frizzled-related family and the Dickkopf (DKK)family. The DKK family currently contains four family members, namelyDKK1 (human DNA accno. NM_(—)012242; PRT accno. 094907), DKK2 (humanaccno. NM_(—)014421; PRT accno. NP_(—)055236), DKK3 (human accno.NM_(—)015881; PRT accno. AAQ88744), and DKK4 (human accno. NM_(—)014420;PRT accno. NP_(—)055235).

Dickkopf-1 (DKK1) is a secreted inhibitor of the Wnt/β-catenin signalingpathway. See, e.g., U.S. patent application 2005-0079173 to Niehrs; U.S.patent application 2004-0234515 to McCarthy. DKK1 possesses the abilityto inhibit Wnt-induced axis duplication, and genetic analysis indicatesthat DKK1 acts upstream to inhibit Wnt signaling. DKK1 is also importantin skeletal development as demonstrated by effects on the loss of bonestructures in chicken and mouse embryos after exposure to elevatedlevels of DKK1 (Tian et al., 2003 New England J. Med. 349:2483-2494).Elevated DKK1 serum levels has been associated with prostate cancer, andelevated DKK1 and RANKL levels in bone marrow plasma and peripheralblood in patients with multiple myeloma are associated with the presenceof focal bone lesions See, e.g., Tian 2003, OMIM accno. 605189. DKK1also plays a role in adipogenesis, chondrogenesis, proliferation of thegastrointestinal epithelial proliferation, bone loss associated withrheumatisms, and initiation of hair follicle placode formation. OMIMaccno. 605189.

Dickkopf-4 (DKK4) is less well characterized but is likewise a secretedinhibitor of the Wnt pathway. DKK4 has been shown to be deposited inplaques in patients with Alzheimers disease and is expressed in muscle.Wnt has no known role in muscle development, therefore it is postulatedherein that DKK4 has an inhibitory role on muscle development.

There is a need for compositions and methods to treat cancers and bonedensity abnormalities, including such agents that interfere orneutralize DKK1 and/or DKK4 mediated antagonism of Wnt signaling.

SUMMARY OF THE INVENTION

An embodiment of the invention herein provides an antibody or an antigenbinding portion thereof that selectively binds to and neutralizes a DKK1and/or a DKK4 polypeptide or a fragment thereof. In a preferredembodiment, the antibody is a DKK1/4 neutralizing antibody. In variousembodiments, the antigen-binding portion of the DKK1/4 neutralizingantibody does not bind a DKK2 or a DKK3.

In one embodiment the antibody or an antigen binding portion thereof isarranged within an immunoglobulin-like scaffold, such as a frameworkselected from, e.g., a human, humanized, humaneered, shark or camelidscaffold, and/or may additionally be recombinant, chimeric, or CDRgrafted antibodies. For instance, technology designed to minimize theHuman Anti-murine Antibody response (humaneering technology of Kalobiosor humanization technology of PDL) are contemplated within theinvention. Further, antigen binding portions specific to DKK1 or DKK4may also be within non-immunoglobulin-like scaffold, including, e.g.,arrayed within an adnectin, fibrinogen, ankyrin-derived repeats, etc.type of framework.

In a particular embodiment, the Dkk1 antibody is characterized as havingan antigen-binding region that is specific for target protein DKK1, andthe antibody or functional fragment binds to DKK1 or a fragment thereof.In a related embodiment, the DKK4 antibody is characterized as having anantigen-binding region that is specific for target protein DKK4, and theantibody or functional fragment binds to DKK4 or a fragment thereof. Ina related embodiment, the Dkk4 antibody is characterized as having anantigen-binding region that is specific for target protein DKK4, and theantibody or functional fragment binds to DKK4 or a fragment thereof. Ina preferred embodiment, the antibody or antigen-binding portion thereofbinds to a DKK1 and a DKK4 polypeptide, but not to a DKK2 or DKK3polypeptide.

In another embodiment the antibody or an antigen binding portion thereofis monoclonal. In another embodiment, the antigen-binding portion ispolyclonal. In various embodiments, the DKK1 antibody or an antigenbinding portion thereof binds a peptide consisting of 30 contiguousamino acids of a DKK1 or a DKK4 polypeptide.

In a related embodiment, the binding to DKK1 or DKK4 is determined atleast by one of the following assays: inhibition of DKK1 or DKK4antagonism of Wnt-signaled transcription; surface plasmon resonanceaffinity determination, enzyme-linked immunosorbent assay binding;electrochemiluminescence-based binding analysis; FMAT, SET, SPR, ALP,TopFlash, blood serum concentration of biomarkers such as osteocalcin(OCN), procollagen type 1 nitrogenous propeptide (P1NP) andosteoprotegrin (OPG), and binding to cell surface receptor(s) such asFrizzled (Fz), LRP (LRP5/6) or Kremen (Krm). In certain embodiments, theDkk1 antibody or antigen-binding portion possesses at least one of thefollowing properties: selectivity for DKK1 that is at least 10³-fold,10⁴-fold or 10⁵-fold greater than for human DKK2 or DKK3; binds to DKK1or DKK4 with a K_(on) of less than 100 nM, 50 nM, 10 nM, 1.0 nM, 500 pM,100 pM, 50 pM or 10 pM; and has an off-rate for DKK1 of less than 10⁻²per sec, 10⁻³ per sec, 10⁻⁴ per sec, or 10⁻⁵ per sec.

In a related embodiment, an antibody of the invention competes with DKK1and/or DKK4 for binding to LRP5/6. In a related embodiment, an antibodyof the invention competes with DKK1 and/or DKK4 for binding to Krm.

In still another embodiment, the invention provides an isolatedantigen-binding region of any of the above antibodies or functionalfragments of these antibodies, and amino acid sequences of these. Thusin certain embodiments, the invention provides isolated amino acidsequences selected from the group of SEQ ID NOs: 2-20 and SEQ ID NOs:40-72 and conservative or humaneered variants of these sequences.

Further, in certain additional embodiments, the invention provides aminoacid sequences SEQ ID NOs: 2-20, 65-72, and conservative or humaneeredvariants of these sequences, which provide isolated antigen-bindingregions each of which is an H-CDR-3.

In a related embodiment, the isolated antigen-binding region is anH-CDR2 region having an amino acid sequence, selected from SEQ ID NOs:2-20, 57-64, and conservative or humaneered variants of these. In stillanother related embodiment, the isolated antigen-binding region is aconsensus H-CDR2 region depicted in sequence selected from the group ofSEQ ID NO: 40 having amino acid sequence GISGSGSYTYYADSVKF, SEQ ID NO:41 having amino acid sequence GISYYGGNTYYADSVKF, and SEQ ID NO: 42having amino acid sequence GISYYGGSTYYADSVKF, and conservative orhumaneered variants of amino acid residues of these sequences.

In certain embodiments, the novel sequence provided is SEQ ID NOs: 2-5,8-11, 20, 49-56, and conservative or humaneered variants of these, whichprovide an isolated antigen-binding region which is an H-CDR1 region.

In a related embodiment, the isolated antigen-binding region is aconsensus H-CDR1 region having an amino acid sequence (using the oneletter amino acid code) selected from amino acid sequences GFTFSSYGMT(SEQ ID NO: 43), GFTFNSYGMT (SEQ ID NO: 44), GFTFSNYGMT (SEQ ID NO: 45),GFTFSSYWMT (SEQ ID NO: 46), GFTFSSYAMT (SEQ ID NO: 47), GFTFSSYGMS (SEQID NO: 48) and conservative or humaneered variants of any of thesesequences.

In another embodiment, the invention provides amino acid sequences whichare isolated antigen-binding regions having an L-CDR3 region, such asamino acid sequences selected from SEQ ID NOs: 21-39, 89-96, andconservative or humaneered variants of these sequences. In still anotherrelated embodiment, the isolated antigen-binding region is an L-CDR1region having an amino acid sequence selected from SEQ ID NOs: 21-39,73-80, and conservative or humaneered variants of these sequences. Inyet another related embodiment, the isolated antigen-binding region isan L-CDR2 region having an amino acid sequence selected from SEQ ID NOs:21-39, 81-88, and conservative or humaneered variants of thesesequences.

In certain embodiments, the isolated antigen-binding region is avariable light chain having an amino acid sequence selected from SEQ IDNOs: 21-39, and conservative or humaneered variants of these sequences.

In another embodiment, the invention provides a nucleotide sequenceselected from the group of SEQ ID NOs: 97-103. In a related embodiment,each of these nucleotide sequences encodes an amino acid sequence andconservative or humaneered variants of these encoded amino acidsequences are also within the scope of the invention herein. In anotherrelated embodiment, the nucleotide sequence of each of SEQ ID NOs:97-100 encodes an antigen binding light chain. In a further relatedembodiment, the nucleotide sequence of each of SEQ ID NOs: 101-103encodes an antigen binding heavy chain. In yet another relatedembodiment, each of these nucleotide sequences is further optimized forexpression in a cell. Preferred cells include, but are not limited to,e.g., Chinese hamster (e.g., CHO) cells, baculovirus, yeast, bacteria,myeloma cells, and/or H. sapiens.

Preferably, sequences are optimized for expression and for productionand clinical use. Characteristics to be optimization for clinical useinclude but are not limited to, e.g., half-life, pharmacokinetics (PK),antigenicity, effector function, FcRn clearance, and patient responseincluding antibody dependent cell cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC) activities.

In a further embodiment, the invention provides an isolatedantigen-binding region having a heavy chain encoded by a nucleotidesequence selected from the group of SEQ ID NOs: 101-103. In a relatedembodiment, the invention provides an isolated antigen-binding regionhaving a light chain encoded by a nucleotide sequence selected from thegroup of SEQ ID NOs: 97-100. In yet another related embodiment, theinvention provides an isolated antigen-binding region having a lightchain encoded by a nucleotide sequence selected from the group of SEQ IDNOs: 97-100, and a heavy chain encoded by a nucleotide sequence selectedfrom the group of SEQ ID NOs: 101-103.

In certain embodiments, the invention provides a nucleotide sequenceselected from the group of SEQ ID NOs: 104-110. In a related embodiment,each of these nucleotide sequences encodes an amino acid sequence andconservative or humaneered variants of these amino acid sequences arealso within the scope of the invention herein. In another relatedembodiment, the nucleotide sequence of each of SEQ ID NOs: 104-107encodes an antigen binding light chain. In a further related embodiment,the nucleotide sequence of each of SEQ ID NOs: 108-110 encodes anantigen binding heavy chain. In yet another related embodiment, each ofthese nucleotide sequences is optimized for expression and/or forclinical use.

In a further embodiment, the invention provides an isolatedantigen-binding region having a heavy chain encoded by a nucleotidesequence selected from the group of SEQ ID NOs: 108-110. In anotherrelated embodiment, the invention provides an isolated antigen-bindingregion having a light chain encoded by a nucleotide sequence selectedfrom the group of SEQ ID NOs: 104-107. In yet another relatedembodiment, the invention provides an isolated antigen-binding regionhaving a light chain encoded by a nucleotide sequence selected from thegroup of SEQ ID NOs: 104-107, and a heavy chain encoded by a nucleotidesequence selected from the group of SEQ ID NOs: 108-110.

In other embodiments, the invention provides an amino acid sequenceselected from the group of SEQ ID NOs: 111-117, and providesconservative variants of these sequences. In a related embodiment, eachof SEQ ID NOs: 111-114 depicts an antigen binding light chain. Inanother embodiment, each of SEQ ID NOs: 115-117 depicts an antigenbinding heavy chain. In yet another related embodiment, the amino acidsequence is optimized for expression and/or for clinical use.

In a further embodiment, the invention provides an isolatedantigen-binding region having a heavy chain depicted in an amino acidsequence selected from the group of SEQ ID NOs: 115-117, and providesconservative variants of these sequences. In a related embodiment, theinvention provides an isolated antigen-binding region having a lightchain depicted in an amino acid sequence selected from the group of SEQID NOs: 111-114, and provides conservative variants of these sequences.In yet another related embodiment, the invention provides an isolatedantigen-binding region having a heavy chain depicted in an amino acidsequence selected from the group of SEQ ID NOs: 115-117, and providesconservative variants of these sequences, and a light chain depicted inan amino acid sequence selected from the group of SEQ ID NOs: 111-114,and provides conservative variants of these sequences.

In another embodiment, the invention provides a nucleotide sequenceselected from the group of SEQ ID NOs: 120-121, and/or the an isolatedantigen-binding region having a variable region of a light chain or aheavy chain encoded by the respective nucleotide sequences. In a relatedembodiment, each of these nucleotide sequences encodes an amino acidsequence, and conservative or humaneered variants of these amino acidsequences are also within the scope of the invention. In another relatedembodiment, the nucleotide sequence of SEQ ID NO: 120 encodes an antigenbinding light chain. In a further related embodiment, the nucleotidesequence of SEQ ID NOs: 121 encodes an antigen binding heavy chain. Inyet another related embodiment, each of these nucleotide sequences isfurther optimized for expression and/or for clinical use.

In certain embodiments, the invention provides an amino acid sequenceselected from the group of 118-119 and conservative or humaneeredvariants of these sequences. In a related embodiment, SEQ ID NO: 118depicts an antigen binding variable region of a light chain. In anotherrelated embodiment, SEQ ID NO: 119 depicts an antigen binding variableregion of a heavy chain. In still another related embodiment, the aminoacid sequence is optimized for expression and/or for clinical use.

In other embodiments, the invention provides an amino acid sequencehaving at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identity with theCDR regions depicted in SEQ ID NOs: 2-39. In a related embodiment, theinvention provides, an amino acid sequence having at least 60, 70, 80,90, 95, 96, 97, 98 or 99% identity with a sequence depicted in SEQ IDNOs: 111-120. In yet another related embodiment, the invention provides,a nucleotide sequence having at least 60, 70, 80, 90, 95, 96, 97, 98 or99% identity with a sequence depicted in SEQ ID NOs: 97-110 and 120-121.

In a certain embodiment, any of the above isolated antibodies is an IgG.In a related embodiment, any of the above isolated antibodies is anIgG1, an IgG2, an IgG3 or an IgG4. In another embodiment, the antibodyis an IgE, an IgM, an IgD or an IgA. In a related embodiment, theinvention is selected from a monoclonal or a polyclonal antibodycomposition. In further embodiments, the antibody is chimeric,humanized, humaneered, recombinant, etc. In yet another embodiment, theinvention provides an isolated human or humanized antibody or functionalfragment of it, having an antigen-binding region that is specific for anepitope of DKK1, and the antibody or functional fragment binds to DKK1or DKK4, or otherwise blocks binding of DKK1 or DKK4 to a cell surfacereceptor (e.g., receptors such as LRP5/6, Kremen, Frizzled). In certainembodiments the antibody or fragment of it prevents, treats, orameliorates development of osteolytic lesions. In other embodiments, theanti-DKK composition of the invention prevents, treats, or ameliorates aDKK1- or DKK4-associated cancer or disease.

In a related embodiment, the invention provides an isolated human orhumanized antibody or functional fragment of it, having anantigen-binding region that is specific for an epitope of target DKK1 orDKK4, and the epitope contains six or more amino acid residues from apolypeptide fragment comprising the CYS1-linker-CYS2 domains of DKK1and/or DKK4. In a related embodiment, the epitope is a conformationalepitope. In a preferred embodiment, the epitope resides within the CYS2domain. In a particular embodiment, the epitope comprises a modifiedamino acid residue. In a related embodiment, the epitope contains atleast one glycosylated amino acid residue.

Functional fragments include Fv and Fab fragments (including singlechain versions such as scFv), as well other antigen-binding regions ofan antibody, including those that are linked to a non-immunoglobulinscaffold and heavy chain antibodies such as camelid and shark antibodiesand nanobodies. In a related embodiment, the isolated antibody asdescribed above is an IgG. In another related embodiment, the isolatedantibody as described above is an IgG1, an IgG2, IgG3 or an IgG4. Inanother embodiment, the antibody is an IgE, an IgM or an IgA. In arelated embodiment, the invention is a polyclonal antibody composition.

In another embodiment, the invention provides a pharmaceuticalcomposition having at least one of any of the above antibodies orfunctional fragments or conservative variants, and a pharmaceuticallyacceptable carrier or excipient of it.

In still another embodiment, the invention provides for a transgenicanimal carrying a gene encoding any of the above antibodies orfunctional fragments of them.

In certain embodiments, the invention provides a method for treating adisorder or condition associated with DKK1 or DKK4 expression. As usedherein, “DKK1-associated diseases” or “DKK4-associated diseases”include, but are not limited to, osteolytic lesions—especiallyosteolytic lesions associated with a myeloma, especially a multiplemyeloma, or with cancers of the bone, breast, colon, melanocytes,hepatocytes, epithelium, esophagus, brain, lung, prostate or pancreas ormetastasis thereof; bone loss associated with transplantation. Furtherdiseases or disorders include but are not limited to, e.g.,osteosarcoma, prostate cancer, hepatocellular carcinoma (HCC), myelomaincluding multiple myeloma, diabetes, obesity, muscle wasting,Alzheimers disease, osteoporosis, osteopenia, rheumatism, colitis and/orunwanted hair loss. The method involves administering to a subject inneed thereof an effective amount of any of the above pharmaceuticalcompositions.

In a related embodiment, the disorder or condition to be treated is abone density abnormality. In another related embodiment, the bonedensity abnormality to be treated is the presence of an osteolyticlesion in the subject.

In another embodiment, the disorder or condition to be treated is anosteoporotic condition, such as an osteolytic lesion associated with acancer. In a related embodiment, the cancer to be treated is a myeloma,such as multiple myeloma, or a cancer of the bone, breast, colon,melanocytes, hepatocytes, epithelium, esophagus, brain, lung, prostateor pancreas or metastasis thereof. In other embodiments, theosteoporotic condition is osteoporosis or osteopenia or osteosarcoma.

In certain embodiments, any of the above methods further involveadministering a chemotherapeutic agent. In a related embodiment, thechemotherapeutic agent is an anti-cancer agent. In another relatedembodiment, the chemotherapeutic agent is an anti-osteoporotic agent.

In still another embodiment, the invention provides a method fortreating a target cell, the method involving blocking DKK1 or DKK4interaction with the cell with any of the above antibodies or functionalfragments of them. In general, the target cell bears a receptor thatbinds DKK1 or DKK4. In one embodiment, the target cell is an osteoblast,wherein treatment with the neutralizing anti-DKK1/4 composition of theinvention will enhance proliferation and stimulate bone formation. Inone embodiment, the target cell is a muscle cell, wherein treatment willcounteract muscle wasting.

In a related embodiment, the method further involves treating a patientwith the target cell with a chemotherapeutic agent or radiation. In arelated embodiment, following administering or contacting, any of themethods above further involves observing amelioration or retardation ofdevelopment of an osteolytic lesion.

In yet another embodiment, the invention provides a method foridentifying DKK1 or DKK4 in serum. This method involves detecting DKK1or DKK4 with any of the above antibodies or antibody fragments furtherhaving a detectable label. The label is radioactive, fluorescent,magnetic, paramagnetic, or chemiluminescent.

In another embodiment, any of the above human or humanized antibodies orantibody fragments are synthetic.

In another embodiment, the invention provides a pharmaceuticalcomposition of any of the above antibodies or functional fragments ofthese antibodies and an additional therapeutic agent. The additionaltherapeutic agent can be selected from the group consisting of ananti-cancer agent; an anti-osteoporotic agent; an antibiotic; anantimetabolic agent; an antidiabetic agent; an anti-inflammatory agent;an anti-angiogenic agent; a growth factor; and a cytokine.

The invention further relates to a method of preventing or treatingproliferative diseases or diseases, such as a cancer, in a mammal,particularly a human, with a combination of pharmaceutical agents whichcomprises:

(a) a DKK1/4 neutralizing agent of the invention; and

(b) one or more pharmaceutically active agents;

wherein at least one pharmaceutically active agent is an anti-cancertherapeutic.

The invention further relates to pharmaceutical compositions comprising:

(a) a DKK1/4 neutralizing antibody;

(b) a pharmaceutically active agent; and

(c) a pharmaceutically acceptable carrier;

wherein at least one pharmaceutically active agent is an anti-cancertherapeutic.

The present invention further relates to a commercial package or productcomprising:

(a) a pharmaceutical formulation of a DKK1/4 neutralizing antibody; and

(b) a pharmaceutical formulation of a pharmaceutically active agent forsimultaneous, concurrent, separate or sequential use;

wherein at least one pharmaceutically active agent is an anti-cancertherapeutic.

In a certain embodiment, the invention provides an antibody having afirst amino acid sequence which is a heavy chain selected from SEQ IDNOs: 2-20, and a sequence having at least 60, 70, 80, 90, 95, 96, 97, 98or 99 percent sequence identity in the CDR regions with the CDR regionshaving SEQ ID NOs: 2-20; and a second amino acid sequence which is alight chain selected from SEQ ID NOs: 21-39, and a sequence having atleast 60, 70, 80, 90, 95, 96, 97, 98 or 99 percent sequence identity inthe CDR regions with the CDR regions shown in SEQ ID NOs: 21-39.

In still another embodiment, the invention provides an immunoconjugatemade out of a first component which is an antibody or fragment asdescribed above and a second component having a second amino acidsequence. For example, the immunoconjugate is a cytotoxin, or theimmunoconjugate is a binding protein or antibody having a bindingspecificity for a target that is different from DKK1 or DKK4. Forexample, the target of the binding specificity different from DKK1 orDKK4 is a tumor antigen or tumor-associated protein on a surface of acancer cell.

In certain embodiments, the invention provides for any of the aboveantibodies to be a bispecific antibody.

In another embodiment, the invention provides a kit having any of theabove antibodies or antibody fragments. In some embodiments, the kitfurther contains a pharmaceutically acceptable carrier or excipient ofit. In other related embodiments, any of the above antibodies in the kitis present in a unit dose. In yet another embodiment, the kit is anELISA diagnostic kit. In a related embodiment, the kit includesinstructions for use in administering any of the above antibodies to asubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating no significant loss of His-Strep-taggedDKK1 from the Strep-Tactin-coated beads when the non-washed beads arecompared with those beads washed with different HuCAL® stringencies.

FIG. 2 is a bar graph illustrating the results of a Wnt activity Assaywith 100 μl Bright-Glo luciferase reagent, as provided in Example 3.

FIG. 3 is a graph of an Enzyme Linked Immuno Sorbent Assay (ELISA). Thefluorescence is measured in a TECAN Spectrafluor plate reader and thebinding curves are shown.

FIG. 4 is a graph illustrating the standard Wnt3a-dependent TCF/LEF lucreporter assay, as provided in Example 6.

FIG. 5 is a graph illustrating an improved version of the TCF/LEF lucreporter assay showing highly improved sensitivity to DKK1 mediated bythe co-expression of the Kremen co-receptor protein.

FIG. 6A is a graphic illustration of the surface plasmin resonancemeasurement of an anti-DKK1/4 antibody binding to DKK1. FIG. 6B is atabulation of calculated binding affinity and kinetic values.

FIG. 7A is a schematic representation of full-length and truncated DKK1for use in epitope mapping. FIG. 7B depicts binding of an antibody ofthe invention to the DKK1 proteins and fragments of FIG. 7A.

FIG. 8 is a graphic illustration of DKK1/4 antibody binding in acompetition ELISA assay.

FIG. 9 is a graphic illustration of DKK1/4 antibody-associatedreactivation of Wnt-regulated gene transcription downstream.

FIG. 10 is a graphic illustration of DKK1/4 antibody reversal DKK1inhibited of ALP secretion.

FIG. 11 is a graphic illustration of DKK1/4 antibody effects on in vivoxenografts.

FIG. 12 is a graphic illustration of DKK1/4 antibody-associatedelevation of bone density in vivo.

FIG. 13 is a graphic illustration comparing anti-osteolytic efficacy ofZometa with a DKK1/4 antibody.

FIG. 14 is a graphic illustration of the dose dependent efficacy of aDKK1/4 antibody on anabolic bone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated DKK1/4 antibodies,particularly human antibodies, that bind specifically to DKK1 or DKK4and that inhibit functional properties of DKK1 or DKK4. In a preferredembodiment, the DKK1/4 antibody does not specifically bind to DKK2 orDKK3. In certain embodiments, the antibodies of the invention arederived from particular heavy and light chain sequences and/or compriseparticular structural features such as CDR regions comprising particularamino acid sequences. The invention provides isolated antibodies,methods of making such antibodies, immunoconjugates and bispecificmolecules comprising such antibodies and pharmaceutical compositionscontaining the antibodies, immunoconjugates or bispecific molecules ofthe invention. The invention also relates to methods of using theantibodies to inhibit a disorder or condition associated DKK1 or DKK4.Contemplated diseases and disorders include, but are not limited to,osteolytic lesions—especially osteolytic lesions associated with amyeloma, especially a multiple myeloma, or with cancers of the bone,breast, colon, melanocytes, hepatocytes, epithelium, esophagus, brain,lung, prostate or pancreas or metastasis thereof; bone loss associatedwith transplantation. Further diseases or disorders include but are notlimited to, e.g., osteosarcoma, prostate cancer, hepatocellularcarcinoma (HCC), myeloma including multiple myeloma, diabetes, obesity,muscle wasting, Alzheimers disease, osteoporosis, osteopenia,rheumatism, colitis and/or unwanted hair loss.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and capable of the transmission of such a signalacross the plasma membrane of a cell. An example of a “cell surfacereceptor” of the present invention is a receptor to which the DKK1 orthe DKK4 protein molecule binds. Such cell surface receptors include,but are not limited to, Frizzled (Fz), LRP (LRP5 and LRP6), and Kremen(Krm).

As used herein, the term “antibody” refers to polyclonal antibodies,monoclonal antibodies, humanized antibodies, single-chain antibodies,and fragments thereof such as F_(ab), F_((ab′)2), F_(v), and otherfragments that retain the antigen binding function of the parentantibody. As such, an antibody may refer to an immunoglobulin orglycoprotein, or fragment or portion thereof, or to a constructcomprising an antigen-binding portion comprised within a modifiedimmunoglobulin-like framework, or to an antigen-binding portioncomprised within a construct comprising a non-immunoglobulin-likeframework or scaffold.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as F_(ab),F_((ab′)2), F_(v), and others that retain the antigen binding functionof the antibody. Monoclonal antibodies of any mammalian species can beused in this invention. In practice, however, the antibodies willtypically be of rat or murine origin because of the availability of rator murine cell lines for use in making the required hybrid cell lines orhybridomas to produce monoclonal antibodies.

As used herein, the term “polyclonal antibody” refers to an antibodycomposition having a heterogeneous antibody population. Polyclonalantibodies are often derived from the pooled serum from immunizedanimals or from selected humans.

As used herein, the phrase “single chain antibodies” refer to antibodiesprepared by determining the binding domains (both heavy and lightchains) of a binding antibody, and supplying a linking moiety whichpermits preservation of the binding function. This forms, in essence, aradically abbreviated antibody, having only that part of the variabledomain necessary for binding to the antigen. Determination andconstruction of single chain antibodies are described in U.S. Pat. No.4,946,778 to Ladner et al.

A “naturally occurring antibody” is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, C_(L). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to the protein sequence that binds thetarget, e.g., one or more CDRs. It includes, e.g., full lengthantibodies, one or more fragments of an antibody, and/or CDRs on anon-immunoglobulin-related scaffold that retain the ability tospecifically bind to an antigen (e.g., DKK1). The antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and CH1domains; a F(ab)_(z) fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR).

As used herein, an “antigen” or an “epitope” interchangeably refer to apolypeptide sequence on a target protein specifically recognized by anantigen-binding portion of an antibody, antibody fragment, or theirequivalents. An antigen or epitope comprises at least 6 amino acids,which may be contiguous within a target sequence, or non-contiguous. Aconformational epitope may comprise non-contiguous residues, andoptionally may contain naturally or synthetically modified amino acidresidues. Modifications to residues include, but are not limited to:phosphorylation, glycosylation, PEGylation, ubiquitinization,furanylization, and the like.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

As described herein, the conservative variants include amino acidresidues in any of the amino acid sequences identified, particularlyconservative changes that are well known to one of ordinary skill in theart of protein engineering.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds DKK1is substantially free of antibodies that specifically bind antigensother than DKK1). An isolated antibody that specifically binds DKK1 may,however, have cross-reactivity to other antigens, such as DKK1 moleculesfrom other species, or other family members such as DKK4 or relatedparalogs. Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies of theinvention may include amino acid residues not encoded by human sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo). However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

As used herein, the term “humanized antibodies” means that at least aportion of the framework regions of an immunoglobulin are derived fromhuman immunoglobulin sequences. A “humanized” antibodies such asantibodies with CDR sequences derived from the germline of anotherspecies, especially a mammalian species, e.g., a mouse, that have beengrafted onto human framework sequences. Example technologies includehumanization technology of PDL.

As used herein, the term “humaneered antibodies” means antibodies thatbind the same epitope but differ in sequence. Example technologiesinclude humaneered antibodies produced by humaneering technology ofKalobios, wherein the sequence of the antigen-binging region is derivedby, e.g., mutation, rather than due to conservative amino acidreplacements.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgA, IgD,IgM, IgE, IgG such as IgG1, IgG2, IgG3 or IgG4) that is provided by theheavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.” As used herein, anantibody that “specifically binds to human DKK1” is intended to refer toan antibody that binds to human DKK1 with a K_(D) of 5×10⁻⁹M or less,2×10⁻⁹M or less, or 1×10⁻¹⁰ M or less. An antibody that “cross-reactswith an antigen other than human DKK1” is intended to refer to anantibody that binds that antigen with a K_(D) of 0.5×10⁻⁸M or less,5×10⁻⁹M or less, or 2×10⁻⁹ M or less. An antibody that “does notcross-react with a particular antigen” is intended to refer to anantibody that binds to that antigen, with a K_(D) of 1.5×10⁻⁸ M orgreater, or a K_(D) of 5-10×10⁻⁸ M or 1×10⁻⁷ M or greater. In certainembodiments, such antibodies that do not cross-react with the antigenexhibit essentially undetectable binding against these proteins instandard binding assays. As used herein, an antibody that “inhibitsbinding of DKK1 to a cell surface receptor” such as LRP, Fz or Krm,refers to an antibody that inhibits DKK1 binding to the receptor with aK of 1 nM or less, 0.75 nM or less, 0.5 nM or less, or 0.25 nM or less.

As used herein, “osteolysis” refers to a decrease in bone density, whichmay be due to various mechanisms of action including, e.g., decreasedosteoblast activity, increased osteoclast activity. Osteolysis thereforeencompasses mechanisms that generically affect bone mineral density. Asused herein, an antibody that “inhibits osteolytic activity” is intendedto refer to an antibody that inhibits loss of bone density either byincreasing bone formation or blocking a bone resorption.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(D),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, by FMAT, or by using abiosensor system such as a Biacore® system.

As used herein, the term “affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

In order to get a higher avidity probe, a dimeric conjugate (twomolecules of JWJ-1 coupled to a FACS marker) can be constructed, thusmaking low affinity interactions (such as with the germline antibody)more readily detected by FACS. In addition, another means to increasethe avidity of antigen binding involves generating dimers or multimersof any of the fibronectin constructs described herein of the DKK1 orDKK4 antibodies. Such multimers may be generated through covalentbinding between individual modules, for example, by imitating thenatural C-to-N-terminus binding or by imitating antibody dimers that areheld together through their constant regions. The bonds engineered intothe Fc/Fc interface may be covalent or non-covalent. In addition,dimerizing or multimerizing partners other than Fc can be used in DKK1or DKK4 hybrids to create such higher order structures.

As used herein, the term “cross-reactivity” refers to an antibody orpopulation of antibodies binding to epitopes on other antigens. This canbe caused either by low avidity or specificity of the antibody or bymultiple distinct antigens having identical or very similar epitopes.Cross reactivity is sometimes desirable when one wants general bindingto a related group of antigens or when attempting cross-species labelingwhen the antigen epitope sequence is not highly conserved in evolution.

As used herein, the term “high affinity” or “high specificity” for anIgG antibody refers to an antibody having a K_(D) of 10⁻⁸M or less,10⁻⁹M or less, or 10⁻¹⁰ M or less for a target antigen. However, “highaffinity” binding can vary for other antibody isotypes. For example,“high affinity” binding for an IgM isotype refers to an antibody havinga K_(D) of 10⁻⁷M or less, or 10⁻⁸M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows chickens, amphibians, reptiles, etc.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, and/or the nucleotidesequence has been altered to remove latent splice donor or spliceacceptor sites. Optimized codon tables are well known in the art for awide variety of species. Sequences for splice donor and acceptor sitesare also known in the art and latent splice sites may be identified,e.g., by analysis of transcript or expression data. Production cellsinclude, but are not limited to, a prokaryotic cell such as e.g., aprokaryotic cell such as a baculovirus or a bacteria (E. coli), or aeukaryotic cell, for example, yeast (e.g., Pichia), a Chinese HamsterOvary cell (CHO), a myeloma cell or a human cell. The optimizednucleotide sequence is engineered to retain completely or as much aspossible the amino acid sequence and residue number originally encodedby the starting nucleotide sequence, which is also known as the“parental” sequence. The optimized sequences herein have been engineeredto have codons that are preferred in the production cells, howeveroptimized expression of these sequences in other eukaryotic andprokaryotic cells is also envisioned herein. The amino acid sequencesencoded by optimized nucleotide sequences are optionally referred to asoptimized.

In related embodiments, polypeptide sequences of neutralizinganti-DKK1/4 compositions of the invention, and the nucleotides thatencode them, are preferably optimized for production and clinical use.Characteristics that may be optimization for clinical use include, butare not limited to, e.g., half-life, pharmacokinetics (PK),antigenicity, effector function, FcRn clearance, and patient responseincluding antibody dependent cell cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC) activities.

As used herein, “DKK1-associated diseases” or “DKK4-associated diseases”include, but are not limited to, osteolytic lesions—especiallyosteolytic lesions associated with a myeloma, especially a multiplemyeloma, or with cancers of the bone, breast, colon, melanocytes,hepatocytes, epithelium, esophagus, brain, lung, prostate or pancreas ormetastasis thereof; bone loss associated with transplantation. Furtherdiseases or disorders include but are not limited to, e.g.,osteosarcoma, prostate cancer, hepatocellular carcinoma (HCC), myelomaincluding multiple myeloma, diabetes, obesity, muscle wasting,Alzheimers disease, osteoporosis, osteopenia, rheumatism, colitis and/orunwanted hair loss.

As used herein, a “treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. The “pathology”of cancer includes all phenomena that compromise the well being of thepatient. This includes, without limitation, abnormal or uncontrollablecell growth, metastasis, interference with the normal functioning ofneighboring cells, release of cytokines or other secretory products atabnormal levels, suppression or aggravation of inflammatory orimmunological response, etc.

Treatment of patients suffering from clinical, biochemical, radiologicalor subjective symptoms of the disease, such as osteolysis, may includealleviating some or all of such symptoms or reducing the predispositionto the disease.

In general, a neutralizing anti-DKK1/4 composition of the inventionprevents, treats, or ameliorates Wnt-related diseases associated withDKK1 or DKK4 or both, but not diseases associated with DKK2, DKK3 orwith other modulators of the Wnt pathway.

Various aspects of the invention are described in further detail in thefollowing subsections.

The Wnt pathway is a major regulator of mesenchymal stem cell (MSC)differentiation into osteoblasts. It is also an important survivalfactor for active osteoblasts. Dickkopf-1 (DKK1) is a Wnt pathwayantagonist expressed predominantly in bone in adults and is upregulatedin myeloma patients with osteolytic lesions. A neutralizing anti-DKK1/4antibody is a truly anabolic agent, which acts through increasingosteoblastic activity while simultaneously decreasing osteoclasticactivity. In contrast, current drugs such as PTH, which are marketed asanabolic agents, in fact increase markers associated with bothosteoblast ands osteoblasts.

Provided in the invention are polyclonal and monoclonal antibodiesselected for binding to DKK1. A preferred antibody has an affinity ofless than 10 pM against human DKK1. In some embodiments, the anti-DKK1antibody crossreacts with DKK4 (Kd˜300 pM) but not DKK2 (affinityanalysis ongoing).

A preferred epitope for an anti-DKK1 or anti-DKK4 antibody is mapped tothe Cys-2 domain (AAs 189-263), which is known to be responsible forboth LRP6 and Kremen binding. In one embodiment, the epitope includes atleast six, and at most thirty, amino acid residues from the Cys-2 domainof a DKK1 or a DKK4 polypeptide. In a certain embodiment, the epitopeincludes a stretch of at least six contiguous amino acids. In anotherembodiments, the preferred binding site is non-linear, i.e., includesnon-contiguous amino acid residues. In some embodiments, binding dependson N-glycosylation. Only one N-glycosylation site is predicted, atresidue 256 in the Cys-2 domain.

In the present invention, a neutralizing anti-DKK1 antibody blocks theinteraction of DKK1 with LRP6 in both ELISA and cell surface bindingassays. As expected this effectively neutralizes the Wnt suppressiveactivity of DKK1 at EC50's below 1 nM in vitro.

In an in vitro model of osteoblast differentiation, mouse 10T1/2 cellsare treated with Wnt3A to stimulate secretion of alkaline phosphatase(AP), a marker for osteoblast activity. DKK1 blocks AP production inthis model and antibodies of the invention fully reverses thisinhibition.

In certain embodiments, an antibody of the invention exhibits doselinear pharmacokinetics (AUC) in mice, with a dose dependent terminalhalf-life of 35-96 hours in mice over a dose of 20-200 μg/mouse.

Using the intratibial model of osteolytic prostate tumor metastasis,antibodies of the invention inhibit tumor-induced cortical bone damage.Effects on trabecular bone are confounded in this model by theobservation that both tumor implants and sham implants cause mechanicaldamage to the bone, which results in an initial increase in woven bonethat is later remodeled, thus causing a decrease in apparent bonevolume. In a certain embodiment, an antibody of the invention increasesthe production of woven bone in both tumor and sham implanted tibias andinhibits the decrease in bone volume accompanying remodeling.

In related embodiments, changes in bone markers (osteocalcin, sRANKL,OPG, AP, TRAP) are used to demonstrate clinical effects of antibodies ofthe invention on bone-related diseases and to predict clinical outcomesof treatments with pharmaceutically effective levels of a neutralizinganti-DKK1 antibody as provided herein. Krm binds DKK in approx sameregion as LRP. Krm needed for Wnt signal inhibition via DKK interactionwith LRP. Krm-DKK-LRP complexed in order to be internalized for Wntpathway deactivation.

A neutralizing anti-DKK1/4 composition of the invention preferably bindsDKK1 or DKK4, blocking their interaction with LRP and/or Krm, therebyneutralizing DKK1 or DKK4 effect on Wnt signaling pathway. Wnt pathwayreactivation occurs only where DKK1 and/or DKK4 are limiting.

Standard assays to evaluate the binding ability of the antibodies towardDKK1 of various species are known in the art, including for example,ELISAs, western blots and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis, BioVeris SET assay, FMAT,chemiluminescense, and/or SPR assays—signal inhibition or releaseassays. Assays to evaluate the effects of the antibodies on functionalproperties of DKK1 are described in further detail in the Examples.

Accordingly, an antibody that “inhibits” one or more of these DKK1functional properties (e.g., biochemical, immunochemical, cellular,physiological or other biological activities, or the like) as determinedaccording to methodologies known to the art and described herein, willbe understood to relate to a statistically significant decrease in theparticular activity relative to that seen in the absence of the antibody(e.g., or when a control antibody of irrelevant specificity is present).An antibody that inhibits DKK1 activity effects such a statisticallysignificant decrease by at least 10% of the measured parameter, by atleast 50%, 80% or 90%, and in certain embodiments an antibody of theinvention may inhibit greater than 95%, 98% or 99% of DKK1 functionalactivity.

Dickkopf Family Members

Appropriate bone metabolism involves a complex web of regulatorypathways and interconnections between osteoblasts, osteoclasts and thesurrounding stroma. Imbalances in these regulatory mechanisms areinvolved in osteolytic conditions such as osteoporosis, tumor-inducedosteolytic lesions, renal and liver transplant induced bone loss, andanti-hormone chemotherapy induced-bone loss. These conditions can havesevere symptoms including bone pain, fractures, vertebral compression,and reduced mobility. Most treatments approved or currently underclinical evaluation act predominantly by inhibiting osteoclast function.For example, bisphosphonates such as zoledronic acid (Zometa) are thecurrent standard of care and act by inhibiting osteoclast function andsurvival. While bisphosphonates are generally effective, some patientsdo not respond robustly or discontinue use due to renal toxicity orosteonecrosis [Markowitz 2003] [Ruggiero 2004]. In addition there aremultiple investigational drugs targeting osteoclast development such asRANKL and M-CSF neutralizing antibodies, or osteoclast function such asCathepsin K inhibitors.

Stimulation of osteoblast activity would provide a novel mechanism bywhich to treat osteolytic disease. The Wnt pathway plays a major role inosteoblast differentiation and activity, while the Dickkopf (DKK) familyof antagonists in particular have been identified as key regulators ofthis process in vitro (see [Krishnan 2006] for review). In vitro, Wnt isan essential osteoblast survival and differentiation factor. Clinicalvalidation for the role of the Wnt pathway is provided by mutations inthe Wnt co-receptor, LRP5. Inactivating mutations in LRP5 result inosteoporosis-pseudoglioma syndrome (OPPG) [Ai 2005], while activatingmutations result in high bone mass [Boyden 2002]. These activatingmutations are found in the extracellular domain and have beendemonstrated to impair binding to the Dickkopf (DKK) family of Wntantagonists.

DKK1 has been reported to be overexpressed in myeloma cells frompatients with bone lesions but absent in normal plasma cells or inplasma cells from patients without bone lesions [Tian 2004] [Politou2006]. Such an overexpression of DKK1 by myeloma cells may upset thenormal balance between osteoblasts and osteoclasts by blockingosteoblast differentiation, and thus promoting bone resorption.Moreover, some of the anti-tumor treatments used for myeloma, such asdexamethasone, have been reported to upregulate DKK1 [Ohnaka 2004].Therefore, an anti-DKK1 neutralizing antibody should allow reactivationof the Wnt pathway in osteolytic lesions, while not affecting Wntsignaling in other tissues where DKK1 levels are relatively low or whereother antagonists predominate. In adults, DKK1 has been reported to behighly expressed only in bone [Li 2006], suggesting an ongoing role forDKK1 in regulating bone metabolism in adults.

Transgenic mice overexpressing Wnt1 in breast tissue develop mammarycarcinomas [Tsukamoto 1988] and −90% of human colorectal cancers havemutations in either APC or β-catenin, two cytoplasmic components of theWnt pathway [Morin 1997] [Rowan 2000]. This type of evidence raisesconcerns that activation of the Wnt pathway could be a risk forincreased tumor initiation or progression. In addition, DKKs are downregulated in colorectal tumors and melanomas, suggesting a potentialtumor suppressive role.

A DKK polypeptide of the invention includes DKK1 (SEQ ID NO:1) and DKK4(SEQ ID NO:124), as well as DKK2 (SEQ ID NO: 122) and DKK3 (SEQ IDNO:123). DKK family members have two CYS domains (CYS1 and CYS2) asshown in the Table A—DKK1 Family Member PileUp. DKK proteins contains anacid N-terminal signal peptide, two CYS domains containing clusters ofcysteine residues separated by a divergent linker region, and apotential C-terminal N-glycosylation site. The CYS2 domain in DKK4 has alipid-binding function that may facilitate WNT/DKK interactions at theplasma membrane. OMIM accno. 605417.

TABLE A DKK1 Family Member PileUp

Antibodies Against DKK1 and DKK4

In a preferred embodiment, the antibody of the invention is specific toa human DKK protein. In a more preferred embodiment, the antibody of theinvention

A DKK1 or DKK4 neutralizing antibody is distinct from the Wnt pathwaymodifications that have been linked to tumor promotion. The Wnt pathwayis regulated by a complex network of extracellular ligands, receptorsand antagonists of which DKK1 is only one. Due to the restrictedexpression of DKK1 in adults and its functional redundancy with otherWnt antagonists a neutralizing DKK1 antibody is unlikely to causewidespread activation of Wnt signaling or therefore, tumorigenesis. Thisis further supported by two observations: first, activating LRP5mutations (inhibiting DKK binding) induce a high bone mass phenotype buthave no apparent increased cancer risk [Moon 2004], while DKK1heterozygous null or Doubleridge mice have decreased DKK1 levels, highbone mass phenotype, but no reported increased rate of tumor formation[MacDonald 2004].

An anti-DKK1 antibody should positively impact myeloma-inducedosteolytic disease while not increasing the risk of de novotumorigenesis. It is expected that such an antibody would be used incombination with anti-tumor chemotherapies and possibly with anti-boneresorption drugs that inhibit osteoclast function.

Polyclonal Antibodies

Antibodies of the invention may be polyclonal antibodies, especiallyhuman polyclonal antibodies. Polyclonals are derived from the pooledserum from immunized animals or from selected humans.

Monoclonal Antibodies

Antibodies of the invention are preferably the human monoclonalantibodies, such as the isolated and structurally characterized, e.g.,in Examples 1-8. Specific V_(H) amino acid sequences of the antibodiesare shown, e.g., in SEQ ID NOs: 2-20. Specific V_(L) amino acidsequences of the antibodies are shown, e.g., in SEQ ID NOs: 21-39.

A V_(H) amino acid sequence of the antibody may be optimized forexpression in a mammalian cell, e.g., such as the sequence shown in SEQID NO: 119. A V_(L) amino acid sequence of the antibodies may beoptimized for expression in a mammalian cell, e.g., such as the sequenceshown in SEQ ID NO: 118. Likewise, sequences may be optimized forexpression in, e.g., yeast, bacteria, hamster and other cells, dependingon which expression system is preferred for the characteristic beingoptimized. Other antibodies of the invention include amino acids thathave been mutated, yet have at least 60, 70, 80, 90, 95, 96, 97, 98 or99 percent identity in the CDR regions with the CDR regions depicted inthe sequences described above.

Further, full length light chain parental nucleotide sequences are shownin SEQ ID NOs: 97-100. Full length heavy chain parental nucleotidesequences are shown in SEQ ID NOs: 101-103. Full length light chainnucleotide sequences optimized for expression in a mammalian cell areshown in SEQ ID NOs: 104-107. Full length heavy chain nucleotidesequences optimized for expression in a mammalian cell are shown in SEQID NOs: 108-110. Full length light chain amino acid sequences encoded byoptimized light chain nucleotide sequences are shown in SEQ ID NOs:111-114. Full length heavy chain amino acid sequences encoded byoptimized heavy chain nucleotide sequences are shown in SEQ ID NOs:115-117. Other antibodies of the invention include amino acids ornucleic acids that have been mutated, yet have at least 60, 70, 80, 90,95, 96, 97, 98 or 99 percent identity to the sequences described above.

Since each of these antibodies can bind to DKK1, the V_(H), V_(L), fulllength light chain, and full length heavy chain sequences (nucleotidesequences and amino acid sequences) can be “mixed and matched” to createother anti-DKK1 binding molecules of the invention. DKK1 binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed above and in the Examples (e.g., ELISAs). When these chainsare mixed and matched, a V_(H) sequence from a particular V_(H)/V_(L)pairing should be replaced with a structurally similar V_(H) sequence.Likewise a full length heavy chain sequence from a particular fulllength heavy chain/full length light chain pairing should be replacedwith a structurally similar full length heavy chain sequence. Likewise,a V_(L) sequence from a particular V_(H)/V_(L) pairing should bereplaced with a structurally similar V_(L) sequence. Likewise a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence. The V_(H), V_(L),full length light chain, and full length heavy chain sequences of theantibodies of the present invention are particularly amenable for mixingand matching, since these antibodies use V_(H), V_(L), full length lightchain, and full length heavy chain sequences derived from the samegermline sequences and thus exhibit structural similarity.

Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody or antigen binding portion thereof having: a V_(H)region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-20 and 119; and a V_(L) region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:21-39 and 118; wherein the antibody specifically binds DKK1.

Examples of heavy and light chain combinations include: a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 2 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 21; or a V_(H) regioncomprising SEQ ID NO: 3 and a V_(L) region comprising SEQ ID NO: 22; ora V_(H) region comprising SEQ ID NO: 4 and a V_(L) region comprising SEQID NO: 23; or a V_(H) region comprising SEQ ID NO: 5 and a V_(L) regioncomprising SEQ ID NO: 24; or a V_(H) region comprising SEQ ID NO: 6 anda V_(L) region comprising SEQ ID NO: 25; or a V_(H) region comprisingSEQ ID NO: 7 and a V_(L) region comprising SEQ ID NO: 28; or a V_(H)region comprising SEQ ID NO: 8 and a V_(L) region comprising SEQ ID NO:29; or a V_(H) region comprising SEQ ID NO: 9 and a V_(L) regioncomprising SEQ ID NO: 30; or a V_(H) region comprising SEQ ID NO: 10 anda V_(L) region comprising SEQ ID NO: 31; or a V_(H) region comprisingSEQ ID NO: 11 and a V_(L) region comprising SEQ ID NO: 32; or a V_(H)region comprising SEQ ID NO: 12 and a V_(L) region comprising SEQ ID NO:33; or a V_(H) region comprising SEQ ID NO: 119 and a V_(L) regioncomprising SEQ ID NO: 118.

In another aspect, the invention provides an isolated monoclonalantibody or antigen binding portion thereof having: a full length heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 115-117; and a full length light chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 111-114.

Thus, examples of full length heavy chain and full length light chaincombinations, respectively, include: SEQ ID NO: 115 with SEQ ID NO: 111;or SEQ ID NO: 116 with SEQ ID NO: 112; or SEQ ID NO: 117 with SEQ ID NO:113; or SEQ ID NO: 117 with SEQ ID NO: 114.

In another aspect, the invention provides an isolated monoclonalantibody or antigen binding portion thereof comprising a full lengthheavy chain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 101-103; and a full length light chain encodedby a nucleotide sequence selected from the group consisting of SEQ IDNOs: 97-100.

Thus, examples of nucleotides that encode full length heavy and lightchains, respectively, that may be combined include: SEQ ID NO: 101 and97; or SEQ ID NO: 102 and 98; or a SEQ ID NO: 103 and 99; or SEQ ID NO:103 and 100.

In yet another aspect, the invention provides an isolated monoclonalantibody or antigen binding portion that has been optimized forexpression in the cell having: a full length heavy chain comprising anucleotide sequence selected from the group consisting of SEQ ID NOs:108-110; and a full length light chain comprising a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 104-107.

In yet another aspect, the invention provides antibodies that comprisethe heavy chain and light chain CDR1s, CDR2s and CDR3s of theantibodies, or combinations thereof. The amino acid sequences of theV_(H) CDR1s of the antibodies are shown in SEQ ID NOs: 2-5, 8-11, 20,and 49-56. The amino acid sequences of the V_(H) CDR2s of the antibodiesand are shown in SEQ ID NOs: 2-20 and 57-64. The amino acid sequences ofthe V_(H) CDR3s of the antibodies are shown in SEQ ID NOs: 2-20 and65-72. The amino acid sequences of the V_(L) CDR1s of the antibodies areshown in SEQ ID NOs: 21-39 and 73-80. The amino acid sequences of theV_(L) CDR2s of the antibodies are shown in SEQ ID NOs: 21-39 and 81-88.The amino acid sequences of the V_(L) CDR3s of the antibodies are shownin SEQ ID NOs: 21-39 and 89-96. The CDR regions are delineated using theKabat system (Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

Given that each of these antibodies can bind to DKK1 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, although each antibody must contain aV_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3 to create other anti-DKK1binding molecules of the invention. DKK1 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISAs). When V_(H) CDR sequences aremixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particularV_(H) sequence should be replaced with a structurally similar CDRsequence(s). Likewise, when V_(L) CDR sequences are mixed and matched,the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L) sequenceshould be replaced with a structurally similar CDR sequence(s).Furthermore, CDR1, CDR2 and/or CDR3 sequence from a particular V_(H) orV_(L) sequence may be specifically or randomly mutated to createantibodies that may be tested for affinity or binding characteristics.It will be readily apparent to the ordinarily skilled artisan that novelV_(H) and V_(L) sequences can be created by substituting one or moreV_(H) and/or V_(L) CDR region sequences with structurally similarsequences from the CDR sequences shown herein for monoclonal antibodiesof the present invention.

An isolated monoclonal antibody, or antigen binding portion thereof has:a V_(H) region CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2-5, 8-11, 20 and 49-56; a V_(H) regionCDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-20 and 57-64; a V_(H) region CDR3 comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:2-20 and 65-72; a V_(L) region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21-39 and 73-80; aV_(L) region CDR2 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 21-39 and 81-88; and a V_(L) region CDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 21-39 and 89-96; wherein the antibody specifically bindsDKK1.

In a certain embodiment, the antibody consists of: a V_(H) region CDR1comprising SEQ ID NO: 2; a V_(H) region CDR2 comprising SEQ ID NO: 6; aV_(H) region CDR3 comprising SEQ ID NO: 7; a V_(L) region CDR1comprising SEQ ID NO: 21; a V_(L) region CDR2 comprising SEQ ID NO: 22;and a V_(L) region CDR3 comprising SEQ ID NO: 23.

In another embodiment, the antibody consists of: a V_(H) region CDR1comprising SEQ ID NO: 3; a V_(H) region CDR2 comprising SEQ ID NO: 12; aV_(H) region CDR3 comprising SEQ ID NO: 13; a V_(L) region CDR1comprising SEQ ID NO: 24; a V_(L) region CDR2 comprising SEQ ID NO: 25;and a V_(L) region CDR3 comprising SEQ ID NO: 26.

In yet another embodiment, the antibody consists of: a V_(H) region CDR1comprising SEQ ID NO: 4; a V_(H) region CDR2 comprising SEQ V_(H) ID NO:14; a V_(H) region CDR3 comprising SEQ ID NO: 15; a V_(L) region CDR1comprising SEQ ID NO: 27; a V_(L) region CDR2 comprising SEQ ID NO: 28;and a V_(L) region CDR3 comprising SEQ ID NO: 29.

In another embodiment, the antibody consists of: a V_(H) region CDR1comprising SEQ ID NO: 5; a V_(H) region CDR2 comprising SEQ ID NO: 16; aV_(H) region CDR3 comprising SEQ ID NO: 17; a V_(L) region CDR1comprising SEQ ID NO: 30; a V_(L) region CDR2 comprising SEQ ID NO: 31;and a V_(L) region CDR3 comprising SEQ ID NO: 32.

In a certain embodiment, the antibody consists of: a V_(H) region CDR1comprising SEQ ID NO: 8; a V_(H) region CDR2 comprising SEQ ID NO: 18; aV_(H) region CDR3 comprising SEQ ID NO: 19; a V_(L) region CDR1comprising SEQ ID NO: 33; a V_(L) region CDR2 comprising SEQ ID NO: 34;and a V_(L) region CDR3 comprising SEQ ID NO: 35.

In another embodiment, the antibody consists of: a V_(H) region CDR1comprising SEQ ID NO: 9; a V_(H) region CDR2 comprising SEQ ID NO: 10; aV_(H) region CDR3 comprising SEQ ID NO: 11; a V_(L) region CDR1comprising SEQ ID NO: V_(H) 36; a V_(L) region CDR2 comprising SEQ IDNO: 37; and a V_(L) region CDR3 comprising SEQ ID NO: 38.

As used herein, a human antibody comprises heavy or V_(L) regions orfull length heavy or light chains that are “the product of” or “derivedfrom” a particular germline sequence if the variable regions or fulllength chains of the antibody are obtained from a system that uses humangermline immunoglobulin genes. Such systems include immunizing atransgenic mouse carrying human immunoglobulin genes with the antigen ofinterest or screening a human immunoglobulin gene library displayed onphage with the antigen of interest. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention has full lengthheavy and light chain amino acid sequences; full length heavy and lightchain nucleotide sequences, variable region heavy and light chainnucleotide sequences, or variable region heavy and light chain aminoacid sequences that are homologous to the amino acid and nucleotidesequences of the antibodies described herein, and wherein the antibodiesretain the desired functional properties of the neutralizing anti-DKK1/4composition of the Invention.

For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a V_(H) region and a V_(L)region, wherein: the V_(H) region comprises an amino acid sequence thatis at least 80% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2-20 and 119; the V_(L) region comprisesan amino acid sequence that is at least 80% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOs: 21-39 and118; the antibody specifically binds to DKK1 and/or DKK4, and theantibody exhibits at least one of the following functional properties:the antibody neutralizes binding of a DKK1 protein to LRP6, Fz and/orKrm, or the antibody neutralizes binding of a DKK4 protein to LRP, Pzand/or Krm.

In a further example, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, comprising a full lengthheavy chain and a full length light chain, wherein: the full lengthheavy chain comprises an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group consistingof SEQ ID NOs: 115-117; the full length light chain comprises an aminoacid sequence that is at least 80% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 111-114; the antibodyspecifically binds to DKK1, and the antibody exhibits at least one ofthe following functional properties: the antibody inhibits binding DKK1protein to the DKK1 receptor or the antibody inhibits DKK1 receptorbinding preventing or ameliorating osteolysis or the antibody inhibitsDKK1 receptor binding preventing or ameliorating osteolytic lesions orthe antibody inhibits DKK1 receptor binding preventing or amelioratingcancer.

In another example, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, comprising a full lengthheavy chain and a full length light chain, wherein: the full lengthheavy chain comprises a nucleotide sequence that is at least 80%homologous to a nucleotide sequence selected from the group consistingof SEQ ID NOs: 101-103; the full length light chain comprises anucleotide sequence that is at least 80% homologous to a nucleotidesequence selected from the group consisting of SEQ ID NOs: 97-100; theantibody specifically binds to DKK1, and the antibody exhibits at leastone of the following functional properties: the antibody inhibitsbinding DKK1 protein to the DKK1 receptor or the antibody inhibits DKK1receptor binding preventing or ameliorating osteolysis or the antibodyinhibits DKK1 receptor binding preventing or ameliorating osteolyticlesions or the antibody inhibits DKK1 receptor binding preventing orameliorating cancer.

In another example, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof that has been optimized forexpression in a cell, comprising a full length heavy chain and a fulllength light chain, wherein: the full length heavy chain comprises anucleotide sequence that is at least 80% homologous to a nucleotidesequence selected from the group consisting of SEQ ID NOs: 108-110; thefull length light chain comprises a nucleotide sequence that is at least80% homologous to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 104-107; the antibody specifically binds toDKK1, and the antibody exhibits at least one of the following functionalproperties: the antibody inhibits binding DKK1 protein to the DKK1receptor or the antibody inhibits DKK1 receptor binding preventing orameliorating osteolysis or the antibody inhibits DKK1 receptor bindingpreventing or ameliorating osteolytic lesions or the antibody inhibitsDKK1 receptor binding preventing or ameliorating cancer.

In another example, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof that has been optimized forexpression in a cell, comprising a V_(H) region and a V_(L) region,wherein: the full length heavy chain comprises a nucleotide sequencethat is at least 80% homologous to a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 121; the full length light chaincomprises a nucleotide sequence that is at least 80% homologous to anucleotide sequence selected from the group consisting of SEQ ID NO:120; the antibody specifically binds to DKK1, and the antibody exhibitsat least one of the following functional properties: the antibodyinhibits binding DKK1 protein to the DKK1 receptor or the antibodyinhibits DKK1 receptor binding preventing or ameliorating osteolysis orthe antibody inhibits DKK1 receptor binding preventing or amelioratingosteolytic lesions or the antibody inhibits DKK1 receptor bindingpreventing or ameliorating cancer.

In various embodiments, the antibody may exhibit one or more, two ormore, or three of the functional properties discussed above. Theantibody can be, for example, a human antibody, a humanized antibody ora chimeric antibody.

As used herein, the percent homology between two amino acid sequences ortwo nucleotide sequences is equivalent to the percent identity betweenthe two sequences. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM 120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al., 1990 J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997 NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http:www.ncbi.nhn.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a V_(H) regionconsisting of CDR1, CDR2, and CDR3 sequences and a V_(L) regionconsisting of CDR1, CDR2, and CDR3 sequences, wherein one or more ofthese CDR sequences have specified amino acid sequences based on theantibodies described herein or conservative modifications thereof, andwherein the antibodies retain the desired functional properties of theneutralizing anti-DKK1/4 composition of the invention. Accordingly, theinvention provides an isolated monoclonal antibody, or antigen bindingportion thereof, consisting of a V_(H) region consisting of CDR1, CDR2,and CDR3 sequences and a V_(L) region consisting of CDR1, CDR2, and CDR3sequences, wherein: the V_(H) regions of CDR1 is sequences consisting ofamino acid sequences selected from the group consisting of amino acidsequences of SEQ ID NOs: 2-5, 8-11, 20, 49-56, and conservativemodifications thereof; the V_(H) region of CDR2 is sequences consistingof amino acid sequences selected from the group consisting of amino acidsequences of SEQ ID NOs: 2-20, 57-64, and conservative modificationsthereof; the V_(H) region of CDR3 is sequences consisting of amino acidsequences selected from the group consisting of amino acid sequences ofSEQ ID NOs: 2-20, 65-72, and conservative modifications thereof; theV_(L) regions of CDR1 is sequences consisting of amino acid sequencesselected from the group consisting of amino acid sequences of SEQ IDNOs: 21-39, 73-80, and conservative modifications thereof; the V_(L)regions of CDR2 is sequences consisting of amino acid sequences selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 21-39,81-88, and conservative modifications thereof; the V_(L) regions of CDR3is sequences consisting of amino acid sequences selected from the groupconsisting of amino acid sequences of SEQ ID NOs: 21-39, 89-96, andconservative modifications thereof; the antibody specifically binds toDKK1; and the antibody exhibits at least one of the following functionalproperties: the antibody inhibits binding DKK1 protein to the DKK1receptor or the antibody inhibits DKK1 receptor binding preventing orameliorating osteolysis or the antibody inhibits DKK1 receptor bindingpreventing or ameliorating osteolytic lesions or the antibody inhibitsDKK1 receptor binding preventing or ameliorating cancer.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

In other embodiments, an antibody of the invention has a full lengthheavy chain sequence and a full length light chain sequence, wherein oneor more of these sequences have specified amino acid sequences based onthe antibodies described herein or conservative modifications thereof,and wherein the antibodies retain the desired functional properties ofthe neutralizing anti-DKK1/4 composition of the invention. Accordingly,the invention provides an isolated monoclonal antibody, or antigenbinding portion thereof, consisting of a full length heavy chain and afull length light chain wherein: the full length heavy chain has aminoacid sequences selected from the group of SEQ ID NOs: 115-117, andconservative modifications thereof; and the full length light chain hasamino acid sequences selected from the group of SEQ ID NOs: 111-114, andconservative modifications thereof; the antibody specifically binds toDKK1; and the antibody exhibits at least one of the following functionalproperties: the antibody inhibits binding DKK1 protein to the DKK1receptor or the antibody inhibits DKK1 receptor binding preventing orameliorating osteolysis or the antibody inhibits DKK1 receptor bindingpreventing or ameliorating osteolytic lesions or the antibody inhibitsDKK1 receptor binding preventing or ameliorating cancer.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

In other embodiments, an antibody of the invention optimized forexpression in a cell has a V_(H) region sequence and a V_(L) regionsequence, wherein one or more of these sequences have specified aminoacid sequences based on the antibodies described herein or conservativemodifications thereof, and wherein the antibodies retain the desiredfunctional properties of the neutralizing anti-DKK1/4 composition of theinvention. Accordingly, the invention provides an isolated monoclonalantibody, or antigen binding portion thereof, consisting of a V_(H)region and a V_(L) region wherein: the V_(H) region has amino acidsequences selected from the group of SEQ ID NO: 119, and conservativemodifications thereof; and the V_(L) region has amino acid sequencesselected from the group of SEQ ID NOs: 118, and conservativemodifications thereof; the antibody specifically binds to DKK1; and theantibody exhibits at least one of the following functional properties:the antibody inhibits binding DKK1 protein to the DKK1 receptor or theantibody inhibits DKK1 receptor binding preventing or amelioratingosteolysis or the antibody inhibits DKK1 receptor binding preventing orameliorating osteolytic lesions or the antibody inhibits DKK1 receptorbinding preventing or ameliorating cancer.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Antibodies that Bind to the Same Epitope as Neutralizing Anti-DKK1/4Composition of the Invention

In another embodiment, the invention provides antibodies that bind tothe same epitope as do the various neutralizing anti-DKK1/4 compositionof the invention provided herein.

Such additional antibodies can be identified based on their ability tocross-compete (e.g., to competitively inhibit the binding of, in astatistically significant manner) with other antibodies of the inventionin standard DKK1 binding assays. The ability of a test antibody toinhibit the binding of antibodies of the present invention to human DKK1demonstrates that the test antibody can compete with that antibody forbinding to human DKK1; such an antibody may, according to non-limitinghypotheses, bind to the same or a related (e.g., a structurally similaror spatially proximal) epitope on human DKK1 as the antibody with whichit competes. In a certain embodiment, the antibody that binds to thesame epitope on human DKK1 as the antibodies of the present invention isa human monoclonal antibody. Such human monoclonal antibodies can beprepared and isolated as described in the Examples.

Camelid and Other Heavy Chain Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as V_(HH) can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. etal., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 BioconjugateChem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineeredlibraries of camelid antibodies and antibody fragments are commerciallyavailable, for example, from Ablynx, Ghent, Belgium. As with otherantibodies of non-human origin, an amino acid sequence of a camelidantibody can be altered recombinantly to obtain a sequence that moreclosely resembles a human sequence, i.e., the nanobody can be“humanized”. Thus the natural low antigenicity of camelid antibodies tohumans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for DKK1. In certain embodiments herein,the camelid antibody or nanobody is naturally produced in the camelidanimal, i.e., is produced by the camelid following immunization withDKK1 or a peptide fragment thereof, using techniques described hereinfor other antibodies. Alternatively, the neutralizing anti-DKK1/4camelid nanobody is engineered, i.e., produced by selection for examplefrom a library of phage displaying appropriately mutagenized camelidnanobody proteins using panning procedures with DKK1 and/or DKK4 as atarget as described in the examples herein. Engineered nanobodies canfurther be customized by genetic engineering to have a half life in arecipient subject of from 45 minutes to two weeks.

In addition to Camelid antibodies, heavy chain antibodies occurnaturally in other animal including but not limited to, e.g., certainspecies of shark and pufferfish (see, e.g., PCT publication WO03/014161). Although variable domains derived from such heavy chainantibodies may be used in the invention, the use of Camelid-derivedheavy chain antibodies and/or of the variable domain sequences thereofis preferred optimization, humanization, humaneering, and the likeand/or for clinical use in humans.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i. e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aV_(H) region comprising CDR1 sequences having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-5, 8-11, 20, 49-56;CDR2 sequences having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-20 and 57-64; CDR3 sequences having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2-20 and65-72, respectively; and a V_(L) region having CDR1 sequences having anamino acid sequence selected from the group consisting of SEQ ID NOs:21-39 and 73-80; CDR2 sequences having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 21-39 and 81-88; and CDR3sequences consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 21-39 and 89-96, respectively. Thus, suchantibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies, yet may contain different framework sequences from theseantibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and V_(L) region genescan be found in the “VBase” human germline sequence database (availableon the Internet at www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E.A., et al., 1991 Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798;and Cox, J. P. L. et al., 1994 Eur. J Immunol. 24:827-836; the contentsof each of which are expressly incorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2and 3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolatedneutralizing anti-DKK1/4 composition, or antigen binding portionsthereof, consisting of a V_(H) region having: a V_(H) CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 2-5, 8-11, 20, 49-56 or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 2-5, 8-11, 20, 49-56; a V_(H) CDR2 region havingan amino acid sequence selected from the group consisting of SEQ ID NOs:2-20 and 57-64, or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NOs: 2-20 and 57-64; a V_(H) CDR3 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 2-20 and65-72, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 2-20 and 65-72; a V_(L) CDR1 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21-39 and 73-80, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 21-39and 73-80; a V_(L) CDR2 region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 21-39 and 81-88, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 21-39and 81-88; and a V_(L) CDR3 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21-39 and 89-96, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 21-39and 89-96.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “back mutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“back mutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “back mutated” antibodiesare also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity.

Furthermore, an antibody of the invention may be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) orbe modified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Each of these embodiments isdescribed in further detail below. The numbering of residues in the Fcregion is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et at.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et at.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret at.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for an “antigen’. Suchcarbohydrate modifications can be accomplished by; for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.The pegylation can be carried out by an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Non-Immunoglobulin Scaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which is specific for the target protein. Such frameworksor scaffolds include the five main idiotypes of human immunoglobulins,or fragments thereof (such as those disclosed elsewhere herein), andinclude immunoglobulins of other animal species, preferably havinghumanized aspects. Single heavy-chain antibodies such as thoseidentified in camelids and/or shark are of particular interest in thisregard. Novel frameworks, scaffolds and fragments continue to bediscovered and developed by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target protein of SEQ ID NO: 1 or SEQ IDNO:122. Such compounds are known herein as “polypeptides comprising atarget-specific binding region”. Known non-immunoglobulin frameworks orscaffolds include, but are not limited to, Adnectins (fibronectin)(Compound Therapeutics, Inc., Waltham, Mass.), ankyrin (MolecularPartners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd(Cambridge, Mass.) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin(Anticalin) (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc. (Mountain View, Calif.)), Protein A (AffibodyAG, Sweden) and affilin (gamma-crystallin or ubiquitin) (Sci1 ProteinsGmbH, Halle, Germany).

(i) Adnectins—Compound Therapeutics

The adnectin scaffolds are based on fibronectin type III domain (e.g.,the tenth module of the fibronectin type III (10 Fn3 domain). Thefibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands. (U.S. Pat. No.6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

(ii) Ankyrin—Molecular Partners

The technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel α-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay.

(iii) Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example,20040175756; 20050053973; 20050048512; and 20060008844.

(vi) Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody®molecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® molecules issimilar to that of an antibody.

(v) Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target molecules of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris Brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is PCT WO 199916873.

(vi) Affilin—Sci1 Proteins

Affilin™ molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New Affilin™ molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulinproteins. Sci1 Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in W0200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Methods of Engineering Antibodies

As discussed above, the anti-DKK1 antibodies having V_(H) and V_(L)sequences or full length heavy and light chain sequences shown hereincan be used to create new anti-DKK1/4 antibodies by modifying fulllength heavy chain and/or light chain sequences, V_(H) and/or V_(L)sequences, or the constant region(s) attached thereto. Thus, in anotheraspect of the invention, the structural features of an anti-DKK1antibody of the invention are used to create structurally relatedanti-DKK1/4 antibodies that retain at least one functional property ofthe antibodies of the invention, such as binding to human DKK1 or DKK4or both and also inhibiting one or more functional properties of DKK1 orDKK4 or both.

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-DKK1 antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-DKK1 antibody consisting of: a V_(H) region antibodysequence having a CDR1 sequence selected from the group consisting ofSEQ ID NOs: 2-5, 8-11, 20, 49-56, a CDR2 sequence selected from thegroup consisting of SEQ ID NOs: 2-20 and 57-64 and/or a CDR3 sequenceselected from the group consisting of SEQ ID NOs: 2-20 and 65-72; and aV_(L) region antibody sequence having a CDR1 sequence selected from thegroup consisting of SEQ ID NOs: 21-39 and 73-80, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 21-39 and 81-88 and/ora CDR3 sequence selected from the group consisting of SEQ ID NOs: 21-39and 89-96; altering at least one amino acid residue within the V_(H)region antibody sequence and/or the V_(L) region antibody sequence tocreate at least one altered antibody sequence; and expressing thealtered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-DKK1 antibody consisting of: a full length heavy chainantibody sequence having a sequence selected from the group of SEQ IDNOs: 115-117; and a full length light chain antibody sequence having asequence selected from the group of SEQ ID NOs: 111-114; altering atleast one amino acid residue within the full length heavy chain antibodysequence and/or the full length light chain antibody sequence to createat least one altered antibody sequence; and expressing the alteredantibody sequence as a protein.

In another embodiment, the invention provides a method for preparing aneutralizing anti-DKK1/4 composition optimized for mammalian expressionconsisting of: a V_(H) region antibody sequence having a sequenceselected from the group of SEQ ID NO: 119; and a V_(L) region antibodysequence having a sequence selected from the group of SEQ ID NO: 118;altering at least one amino acid residue within the V_(H) regionantibody sequence and/or the V_(L) region antibody sequence to create atleast one altered antibody sequence; and expressing the altered antibodysequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the neutralizing anti-DKK1/4 compositionsdescribed herein, which functional properties include, but are notlimited to, specifically binding to human DKK1; and the antibodyexhibits at least one of the following functional properties: theantibody inhibits binding of DKK1 protein to the DKK1 receptor, or theantibody inhibits DKK1 receptor binding preventing or amelioratingosteolysis, or the antibody inhibits DKK1 receptor binding therebypreventing or ameliorating osteolytic lesions, or the antibody inhibitsDKK1 receptor binding preventing or ameliorating cancer.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-DKK1 antibody coding sequence and the resultingmodified anti-DKK1 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

The Fc constant region of an antibody is critical for determining serumhalf-life and effector functions, i.e., antibody dependent cellcytotoxicity (ADCC) or complement dependent cytotoxicity (CDC)activities. One can engineer specific mutants of the Fc fragment toalter the effector function and/or serum half-life (see, e.g., Xencortechnology, see also, e.g., WO2004029207).

One method to alter effector function and serum half-life of an antibodyis to graft the variable region of an antibody fragment with an Fcfragment having the appropriate effector function. IgG1 or IgG4 isotypescan be selected for cell killing activity, whereas IgG2 isotype can beused for silent or neutralizing antibodies (with no cell killingactivity).

Silent antibodies with long serum half-life can be obtained by makingchimeric fusion of variable regions of an antibody with a serum proteinsuch as HSA or a protein binding to such serum protein, such HSA-bindingprotein.

Effector functions can also be altered by modulating the glycosylationpattern of the antibody. Glycart (e.g., U.S. Pat. No. 6,602,684), Biowa(e.g., U.S. Pat. No. 6,946,292) and Genentech (e.g WO03/035835) haveengineered mammalian cell lines to produce antibodies with increased ordecreased effector function. Especially, non fucosylated antibodies willhave enhanced ADCC activities. Glycofi has also developed yeast celllines capable of producing specific glycoforms of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. Examples of full length lightchain parental nucleotide sequences are shown in SEQ ID NOs: 97-100.Examples of full length heavy chain parental nucleotide sequences areshown in SEQ ID NOs: 101-103. Examples of full length light chainnucleotide sequences optimized for expression in a cell are shown in SEQID NOs: 104-107. Examples of full length heavy chain nucleotidesequences optimized for expression in a cell are shown in SEQ ID NOs:108-110.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of theinvention can be, for example, DNA or RNA and may or may not containintronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromvarious phage clones that are members of the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to an scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA molecule, or to a fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked”, as used in this context, isintended to mean that the two DNA fragments are joined in a functionalmanner, for example, such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame, or such that the protein is expressedunder control of a desired promoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, an IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragmentheavy chain gene, the V_(H)-encoding DNA can be operatively linked toanother DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991 Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., 1988 Science 242:423-426; Huston et al., 1988 Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstDKK1 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM mice, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et al., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchromosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-DKK1 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-DKK1 antibodies of the invention. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise anti-DKK1antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Human antibody libraries screens may be used to identify an antibody ofthe invention. Choice of screening technologies include, but are notlimited to, e.g. phage display (Morphosys), the type of libraries (e.g.,HuCal library from Morphosys), affinity maturation technology andfurther codon optimization sequence.

Generation of Human Monoclonal Antibodies Against DKK1.

Purified recombinant human DKK1 derived from E. coli, baculovirus orHEK-EBNA cells, or purified recombinant human DKK1 conjugated to keyholelimpet hemocyanin (KLH), is used as the antigen.

Fully human monoclonal antibodies to DKK1 are prepared using HCo7, HCo12and HCo17 strains of HuMab transgenic mice and the KM strain oftransgenic transchromosomic mice, each of which express human antibodygenes. In each of these mouse strains, the endogenous mouse kappa lightchain gene can be homozygously disrupted as described in Chen et al.,1993 EMBO J. 12:811-820 and the endogenous mouse heavy chain gene can behomozygously disrupted as described in Example 1 of PCT Publication WO01109187. Each of these mouse strains carries a human kappa light chaintransgene, KCo5, as described in Fishwild et al., 1996 NatureBiotechnology 14:845-851. The HCo7 strain carries the HCo7 human heavychain transgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and5,545,807. The HCo12 strain carries the HCo12 human heavy chaintransgene as described in Example 2 of PCT Publication WO 01/09187. TheHCo17 stain carries the HCo17 human heavy chain transgene. The KNMstrain contains the SC20 transchromosome as described in PCT PublicationWO 02/43478.

To generate fully human monoclonal antibodies to DKK1, HuMab mice and KMmice are immunized with purified recombinant DKK1 derived from E. colior DKK1-KLH conjugate as antigen. General immunization schemes for HuMabmice are described in Lonberg, N. et al., 1994 Nature 368(6474):856-859; Fishwild, D. et al., 1996 Nature Biotechnology 14:845-851 andPCT Publication WO 98/24884. The mice are 6-16 weeks of age upon thefirst infusion of antigen. A purified recombinant preparation (5-50 μg)of DKK1 antigen (e.g., purified from transfected E. coli cellsexpressing DKK1) is used to immunize the HuMab mice and KM miceintraperitonealy, subcutaneously (Sc) or by footpad injection.

Transgenic mice are immunized twice with antigen in complete Freund'sadjuvant or Ribi adjuvant either intraperitonealy (IP), subcutaneously(Sc) or by footpad (FP), followed by 3-21 days IP, Sc or FP immunization(up to a total of 11 immunizations) with the antigen in incompleteFreund's or Ribi adjuvant. The immune response is monitored byretroorbital bleeds. The plasma is screened by ELISA, and mice withsufficient titers of anti-DKK1 human immunogolobulin are used forfusions. Mice are boosted intravenously with antigen 3 and 2 days beforesacrifice and removal of the spleen. Typically, 10-35 fusions for eachantigen are performed. Several dozen mice are immunized for eachantigen. A total of 82 mice of the HCo7, HCo12, HCo17 and KM micestrains are immunized with DKK1.

To select HuMab or KM mice producing antibodies that bound DKK1, serafrom immunized mice can be tested by ELISA as described by Fishwild, D.et al., 1996. Briefly, microtiter plates are coated with purifiedrecombinant DKK1 from E. coli at 1-2 μg/ml in PBS, 50 μl/wells incubated4° C. overnight then blocked with 200 μl/well of 5% chicken serum inPBS/Tween (0.05%). Dilutions of plasma from DKK1-immunized mice areadded to each well and incubated for 1-2 hours at ambient temperature.The plates are washed with PBS/Tween and then incubated with agoat-anti-human IgG Fc polyclonal antibody conjugated with horseradishperoxidase (HRP) for 1 hour at room temperature. After washing, theplates are developed with ABTS substrate (Sigma, A-1888, 0.22 mg/ml) andanalyzed by spectrophotometer at OD 415-495. Mice that developed thehighest titers of anti-DKK1 antibodies are used for fusions. Fusions areperformed and hybridoma supernatants are tested for anti-DKK1 activityby ELISA.

The mouse splenocytes, isolated from the HuMab mice and KM mice, arefused with PEG to a mouse myeloma cell line based upon standardprotocols. The resulting hybridomas are then screened for the productionof antigen-specific antibodies. Single cell suspensions of spleniclymphocytes from immunized mice are fused to one-fourth the number ofSP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG(Sigma). Cells are plated at approximately 1×10⁵/well in flat bottommicrotiter plate, followed by about two week incubation in selectivemedium containing 10% fetal bovine serum, 10% P388D 1(ATCC, CRL TIB-63)conditioned medium, 3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013,with high glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES,0.055 mM 2-mercaptoethanol, 50 mglnni gentamycin and 1×HAT (Sigma, CRLP-7185). After 1-2 weeks, cells are cultured in medium in which the HATis replaced with HT. Individual wells are then screened by ELISA forhuman anti-DKK1 monoclonal IgG antibodies. Once extensive hybridomagrowth occurred, medium is monitored usually after 10-14 days. Theantibody secreting hybridomas are replated, screened again and, if stillpositive for human IgG, anti-DKK1 monoclonal antibodies are subcloned atleast twice by limiting dilution. The stable subclones are then culturedin vitro to generate small amounts of antibody in tissue culture mediumfor further characterization.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of the invention,such mice can be immunized with a purified or enriched preparation ofDKK1 antigen and/or recombinant DKK1, or an DKK1 fusion protein, asdescribed by Lonberg, N. et al., 1994 Nature 368(6474): 856-859;Fishwild, D. et al., 1996 Nature Biotechnology 14: 845-851; and PCTPublication. WO 98124884 and WO 01/14424. The mice can be 6-16 weeks ofage upon the first infusion. For example, a purified or recombinantpreparation (5-50 μg) of DKK1 antigen can be used to immunize the humanIg mice intraperitoneally.

Detailed procedures to generate fully human monoclonal antibodies toDKK1 are described above. Cumulative experience with various antigenshas shown that the transgenic mice respond when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by every other week IP immunizations (up to a total of 6) withantigen in incomplete Freund's adjuvant. However, adjuvants other thanFreund's are also found to be effective. In addition, whole cells in theabsence of adjuvant are found to be highly immunogenic. The immuneresponse can be monitored over the course of the immunization protocolwith plasma samples being obtained by retroorbital bleeds. The plasmacan be screened by ELISA, and mice with sufficient titers of anti-DKK1human immunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen. It is expected that 2-3 fusions for each immunization may needto be performed. Between 6 and 24 mice are typically immunized for eachantigen. Usually both HCo7 and HCo12 strains are used. In addition, bothHCo7 and HCo 12 transgene can be bred together into a single mousehaving two different human heavy chain transgenes (HCo7/HCo12).

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×145in flat bottom microtiter plates, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0:055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −a 80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and V_(H) regions of theantibodies described herein can be used to create full-length antibodygenes of any antibody isotype by inserting them into expression vectorsalready encoding heavy chain constant and light chain constant regionsof the desired isotype such that the V_(H) segment is operatively linkedto the CH segment(s) within the vector and the V_(L) segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, inparticular mammalian host cells, is discussed because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded and antigenbinding antibody. Prokaryotic expression of antibody genes has beenreported to be ineffective for production of high yields of activeantibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today 6:12-13).

Mammalian host cells for expressing the recombinant antibodies of theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P.A. Sharp, 1982 Mol. Biol. 159:601-621, NSO myelomacells, COS cells and SP2 cells. In particular, for use with NSO myelomacells, another expression system is the GS gene expression system shownin WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination of aneutralizing anti-DKK1/4 composition, or antigen-binding portion(s)thereof, of the present invention, formulated together with apharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) antibodies, orimmunoconjugates or bispecific molecules of the invention. For example,a pharmaceutical composition of the invention can comprise a combinationof antibodies (or immunoconjugates or bispecifics) that bind todifferent epitopes on the target antigen or that have complementaryactivities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents.

For example, the combination therapy can include an anti-DKK1 antibodyof the present invention combined with at least one otheranti-inflammatory or anti-osteoprotic agent. Examples of therapeuticagents that can be used in combination therapy are described in greaterdetail below in the section on uses of the antibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al., 1977 J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, one can include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption for example, monostearatesalts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 per centto about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 per cent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 200 mg/kg, and more usually 0.01 to 50 mg/kg, of the host bodyweight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-20 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Dosage regimens for an anti-DKK1 antibody ofthe invention include 1 mg/kg body weight or 3 mg/kg body weight byintravenous administration, with the antibody being given using one ofthe following dosing schedules: every four weeks for six dosages, thenevery three months; every three weeks; 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring patient blood levels of antibody to the target antigen or ofsome biomarker such as OCN, OPG or P1NP. In some methods, dosage isadjusted to achieve a plasma antibody concentration of about 1-1000μg/ml and in some methods about 25-300 μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-DKK1 antibody of theinvention can results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.

A composition of the present invention can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the inventioninclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrastemal injection andinfusion.

Alternatively, an antibody of the invention can be administered by anonparenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices shown in U.S. Pat. No. 5,399,163; 5,383,851;5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples ofwell known implants and modules useful in the present invention include:U.S. Pat. No. 4,487,603, which shows an implantable micro-infusion pumpfor dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which shows a therapeutic device for administering medicants through theskin; U.S. Pat. No. 4,447,233, which shows a medication infusion pumpfor delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which shows a variable flow implantable infusion apparatusfor continuous drug delivery; U.S. Pat. No. 4,439,196, which shows anosmotic drug delivery system having multi-chamber compartments; and U.S.Pat. No. 4,475,196, which shows an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade,1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M.Owais et al., 1995 Antimicrob. Agents Chernother. 39:180); surfactantprotein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120(Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen, 1994 FEBS Lett. 346:123; J. J. Killion; I. J. Fidler,1994 Immunomethods 4:273.

The Combinations

The invention further relates to a method of preventing or treatingproliferative diseases or diseases, such as a cancer, in a mammal,particularly a human, with a combination of pharmaceutical agents whichcomprises

(a) a neutralizing anti-DKK1/4 composition; and

(b) one or more pharmaceutically active agents.

The invention further relates to pharmaceutical compositions comprising:

(a) a neutralizing anti-DKK1/4 composition;

(b) a pharmaceutically active agent; and

(c) a pharmaceutically acceptable carrier.

The present invention further relates to a commercial package or productcomprising:

(a) a pharmaceutical formulation of a neutralizing anti-DKK1/4composition; and

(b) a pharmaceutical formulation of a pharmaceutically active agent forsimultaneous, concurrent, separate or sequential use.

The Pharmaceutically Active Agents

The term “pharmaceutically active agents” is a broad one covering manypharmaceutically active agents having different mechanisms of action.Combinations of some of these with DKK1/4 neutralizingantibodies/compositions can result in improvements in cancer therapy.Generally, pharmaceutically active agents are classified according tothe mechanism of action. Many of the available agents areanti-metabolites of development pathways of various tumors, or reactwith the DNA of the tumor cells. There are also agents which inhibitenzymes, such as topoisomerase I and topoisomerase II, or which areanti-mitotic agents.

By the term “pharmaceutically active agent” is meant especially anypharmaceutically active agent other than a neutralizing anti-DKK1/4composition or a derivative thereof. It includes, but is not limited to:

i. an aromatase inhibitor;

ii. an anti-estrogen, an anti-androgen or a gonadorelin agonist;

iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor;

iv. a microtubule active agent, an alkylating agent, an anti-neoplasticanti-metabolite or a platin compound;

v. a compound targeting/decreasing a protein or lipid kinase activity ora protein or lipid phosphatase activity, a further anti-angiogeniccompound or a compound which induces cell differentiation processes;

vi. monoclonal antibodies;

vii. a cyclooxygenase inhibitor, a bisphosphonate, a heparanaseinhibitor, a biological response modifier;

viii. an inhibitor of Ras oncogenic isoforms;

ix. a telomerase inhibitor;

x. a protease inhibitor, a matrix metalloproteinase inhibitor, amethionine aminopeptidase inhibitor, or a proteasome inhibitor;

xi. agents used in the treatment of hematologic malignancies orcompounds which target, decrease or inhibit the activity of Flt-3;

xii. an HSP90 inhibitor;

xiii. antiproliferative antibodies;

xiv. a histone deacetylase (HDAC) inhibitor;

xv. a compound which targets, decreases or inhibits theactivity/function of serine/threonine mTOR kinase;

xvi. a somatostatin receptor antagonist;

xvii. an anti-leukemic compound;

xviii. tumor cell damaging approaches;

xix. an EDG binder;

xx. a ribonucleotide reductase inhibitor;

xxi. an S-adenosylmethionine decarboxylase inhibitor;

xxii. a monoclonal antibody of VEGF or VEGFR;

xxiii. photodynamic therapy;

xxiv. an angiostatic steroid;

xxv. an implant containing corticosteroids;

xxvi. an AT1 receptor antagonist; and

xxvii. an ACE inhibitor.

The term “aromatase inhibitor”, as used herein, relates to a compoundwhich inhibits the estrogen production, i.e., the conversion of thesubstrates androstenedione and testosterone to estrone and estradiol,respectively. The term includes, but is not limited to, steroids,especially atamestane, exemestane and formestane; and, in particular,non-steroids, especially aminoglutethimide, roglethimide,pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole,fadrozole, anastrozole and letrozole. Exemestane is marketed asAROMASIN; formestane as LENTARON; fadrozole as AFEMA; anastrozole asARIMIDEX; letrozole as FEMARA or FEMAR; and aminoglutethimide asORIMETEN. A combination of the invention comprising a pharmaceuticallyactive agent which is an aromatase inhibitor is particularly useful forthe treatment of hormone receptor positive tumors, e.g., breast tumors.

The term “anti-estrogen”, as used herein, relates to a compound whichantagonizes the effect of estrogens at the estrogen receptor level. Theterm includes, but is not limited to, tamoxifen, fulvestrant, raloxifeneand raloxifene hydrochloride. Tamoxifen can be administered in the formas it is marketed, e.g., NOLVADEX; and raloxifene hydrochloride ismarketed as EVISTA. Fulvestrant can be formulated as disclosed in U.S.Pat. No. 4,659,516 and is marketed as FASLODEX. A combination of theinvention comprising a pharmaceutically active agent which is ananti-estrogen is particularly useful for the treatment of estrogenreceptor positive tumors, e.g., breast tumors.

The term “anti-androgen”, as used herein, relates to any substance whichis capable of inhibiting the biological effects of androgenic hormonesand includes, but is not limited to, bicalutamide (CASODEX), which canbe formulated, e.g., as disclosed in U.S. Pat. No. 4,636,505.

The term “gonadorelin agonist”, as used herein, includes, but is notlimited to, abarelix, goserelin and goserelin acetate. Goserelin isdisclosed in U.S. Pat. No. 4,100,274 and is marketed as ZOLADEX.Abarelix can be formulated, e.g., as disclosed in U.S. Pat. No.5,843,901.

The term “topoisomerase I inhibitor”, as used herein, includes, but isnot limited to, topotecan, gimatecan, irinotecan, camptothecian and itsanalogues, 9-nitrocamptothecin and the macromolecular camptothecinconjugate PNU-166148 (compound Al in WO 99/17804). Irinotecan can beadministered, e.g., in the form as it is marketed, e.g., under thetrademark CAMPTOSAR. Topotecan can be administered, e.g., in the form asit is marketed, e.g., under the trademark HYCAMTIN.

The term “topoisomerase II inhibitor”, as used herein, includes, but isnot limited to, the anthracyclines, such as doxorubicin, includingliposomal formulation, e.g., CAELYX, daunorubicin, including liposomalformulation, e.g., DAUNOSOME, epirubicin, idarubicin and nemorubicin;the anthraquinones mitoxantrone and losoxantrone; and thepodophillotoxines etoposide and teniposide. Etoposide is marketed asETOPOPHOS; teniposide as VM 26-BRISTOL; doxorubicin as ADRIBLASTIN orADRIAMYCIN; epirubicin as FARMORUBICIN; idarubicin as ZAVEDOS; andmitoxantrone as NOVANTRON.

The term “microtubule active agent” as used herein, relates tomicrotubule stabilizing, microtubule destabilizing agents andmicrotublin polymerization inhibitors including, but not limited to,taxanes, e.g., paclitaxel and docetaxel; vinca alkaloids, e.g.,vinblastine, especially vinblastine sulfate; vincristine, especiallyvincristine sulfate and vinorelbine; discodermolides; cochicine andepothilonesand derivatives thereof, e.g., epothilone B or a derivativethereof. Paclitaxel is marketed as TAXOL; docetaxel as TAXOTERE;vinblastine sulfate as VINBLASTIN R.P; and vincristine sulfate asFARMISTIN. Also included are the generic forms of paclitaxel as well asvarious dosage forms of paclitaxel. Generic forms of paclitaxel include,but are not limited to, betaxolol hydrochloride. Various dosage forms ofpaclitaxel include, but are not limited to albumin nanoparticlepaclitaxel marketed as ABRAXANE; ONXOL, CYTOTAX Discodermolide can beobtained, e.g., as disclosed in U.S. Pat. No. 5,010,099. Also includedare Epotholine derivatives which are disclosed in U.S. Pat. No.6,194,181, WO 98/10121, WO 98/25929, WO 98/08849, WO 99/43653, WO98/22461 and WO 00/31247. Especially preferred are Epotholine A and/orB.

The term “alkylating agent”, as used herein, includes, but is notlimited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNUor Gliadel), or temozolamide (TEMODAR).Cyclophosphamide can beadministered, e.g., in the form as it is marketed, e.g., under thetrademark CYCLOSTIN; and ifosfamide as HOLOXAN.

The term “anti-neoplastic anti-metabolite” includes, but is not limitedto, 5-fluorouracil (5-FU); capecitabine; gemcitabine; DNA de-methylatingagents, such as 5-azacytidine and decitabine; methotrexate; edatrexate;and folic acid antagonists such as, but not limited to, pemetrexed.Capecitabine can be administered, e.g., in the form as it is marketed,e.g., under the trademark XELODA; and gemcitabine as GEMZAR.

The term “platin compound”, as used herein, includes, but is not limitedto, carboplatin, cis-platin, cisplatinum, oxaliplatin, Satraplatin andplatinum agents such as ZD0473. Carboplatin can be administered, e.g.,in the form as it is marketed, e.g., CARBOPLAT; and oxaliplatin asELOXATIN.

The term “compounds targeting/decreasing a protein or lipid kinaseactivity; or a protein or lipid phosphatase activity; or furtheranti-angiogenic compounds”, as used herein, includes, but is not limitedto, protein tyrosine kinase and/or serine and/or theroine kinaseinhibitors or lipid kinase inhibitors, for example:

i) compounds targeting, decreasing or inhibiting the activity of thevascular endothelial growth factor-receptors (VEGF), such as compoundswhich target, decrease or inhibit the activity of VEGF, especiallycompounds which inhibit the VEGF receptor, such as, but not limited to,7H-pyrrolo[2,3-d]pyrimidine derivatives (AEE788); BAY 43-9006;isolcholine compounds disclosed in WO 00/09495 such as(4-tert-butyl-phenyl)-94-pyridin-4-ylmethyl-isoquinolin-1-yl)-amine(AAL881); and

ii) compounds targeting, decreasing or inhibiting the activity of theplatelet-derived growth factor-receptors (PDGFR), such as compoundswhich target, decrease or inhibit the activity of PDGFR, especiallycompounds which inhibit the PDGF receptor, e.g., aN-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, SU101, SU6668and GFB-111;

iii) compounds targeting, decreasing or inhibiting the activity of thefibroblast growth factor-receptors (FGFR);

iv) compounds targeting, decreasing or inhibiting the activity of theinsulin-like growth factor receptor 1 (IGF-1R), such as compounds whichtarget, decrease or inhibit the activity of IGF-IR, especially compoundswhich inhibit the IGF-1R receptor. Compounds include but are not limitedto the compounds disclosed in WO 02/092599 and derivatives thereof of4-amino-5-phenyl-7-cyclobutyl-pyrrolo[2,3-d]pyrimidine derivatives(AEW541);

v) compounds targeting, decreasing or inhibiting the activity of the Trkreceptor tyrosine kinase family;

vi) compounds targeting, decreasing or inhibiting the activity of theAxl receptor tyrosine kinase family;

vii) compounds targeting, decreasing or inhibiting the activity of thec-Met receptor;

viii) compounds targeting, decreasing or inhibiting the activity of theRet receptor tyrosine kinase;

ix) compounds targeting, decreasing or inhibiting the activity of theKit/SCFR receptor tyrosine kinase;

x) compounds targeting, decreasing or inhibiting the activity of theC-kit receptor tyrosine kinases (part of the PDGFR family), such ascompounds which target, decrease or inhibit the activity of the c-Kitreceptor tyrosine kinase family, especially compounds which inhibit thec-Kit receptor, e.g., imatinib;

xi) compounds targeting, decreasing or inhibiting the activity ofmembers of the c Ab1 family and their gene-fusion products, e.g.,BCR-Ab1 kinase, such as compounds which target decrease or inhibit theactivity of c-AbI family members and their gene fusion products, e.g., aN-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, PD180970, AG957,NSC 680410 or PD173955 from ParkeDavis; BMS354825

xii) compounds targeting, decreasing or inhibiting the activity ofmembers of the protein kinase C (PKC) and Raf family of serine/threoninekinases, members of the MEK, SRC, JAK, FAK, PDK and Ras/MAPK familymembers, or PI(3) kinase family, or of the PI(3)-kinase-related kinasefamily, and/or members of the cyclin-dependent kinase family (CDK) andare especially those staurosporine derivatives disclosed in U.S. Pat.No. 5,093,330, e.g., midostaurin; examples of further compounds include,e.g., UCN-01; safingol; BAY 43-9006; Bryostatin 1; Perifosine;Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521;LY333531/LY379196; isochinoline compounds, such as those disclosed in WO00/09495; FTIs; PD184352 or QAN697, a P13K inhibitor;

xiii) compounds targeting, decreasing or inhibiting the activity ofprotein-tyrosine kinase, such as imatinib mesylate (GLEEVEC); tyrphostinor pyrymidylaminobenzamide and derivatives thereof (AMN107). Atyrphostin is preferably a low molecular weight (Mr<1500) compound, or apharmaceutically acceptable salt thereof, especially a compound selectedfrom the benzylidenemalonitrile class or the S arylbenzenemalonirile orbisubstrate quinoline class of compounds, more especially any compoundselected from the group consisting of Tyrphostin A23/RG-50810, AG 99,Tyrphostin AG 213, Tyrphostin AG 1748, Tyrphostin AG 490, TyrphostinB44, Tyrphostin B44 (+) enantiomer, Tyrphostin AG 555, AG 494,Tyrphostin AG 556; AG957 and adaphostin(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester,NSC 680410, adaphostin);

xiv) compounds targeting, decreasing or inhibiting the activity of theepidermal growth factor family of receptor tyrosine kinases (EGFR,ErbB2, ErbB3, ErbB4 as homo- or heterodimers), such as compounds whichtarget, decrease or inhibit the activity of the epidermal growth factorreceptor family are especially compounds, proteins or antibodies whichinhibit members of the EGF receptor tyrosine kinase family, e.g., EGFreceptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF-related ligands,and are in particular those compounds, proteins or monoclonal antibodiesgenerically and specifically disclosed in WO 97/02266, e.g., thecompound of Example 39, or in EP 0 564 409, WO 99/03854, EP 0520722, EP0 566 226, EP 0 787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO98/10767, WO 97/30034, WO 97/49688, WO 97/38983 and, especially, WO96/30347, e.g., compound known as CP 358774, WO 96/33980, e.g., compoundZD 1839; and WO 95/03283, e.g., compound ZM105180, e.g., trastuzumab(HERCEPTIN®), cetuximab, Iressa, OSI-774, CI 1033, EKB-569, GW-2016,E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and7H-pyrrolo-[2,3-d]pyrimidine derivatives which are disclosed in WO03/013541, erlotinib and gefitinib. Erlotinib can be administered in theform as it is marketed, e.g. TARCEVA, and gefitinib as IRESSA, humanmonoclonal antibodies against the epidermal growth factor receptorincluding ABX-EGFR; and

xv) Compounds which target, decrease or inhibit the activity/function ofserine/theronine mTOR kinase are especially compounds, proteins orantibodies which target/inhibit members of the mTOR kinase family, e.g.,RAD, RAD001, CCI-779, ABT578, SAR543, rapamycin and derivatives/analogsthereof, AP23573 and AP23841 from Ariad, everolimus (CERTICAN) andsirolimus. CERTICAN (everolimus, RAD) an investigational novelproliferation signal inhibitor that prevents proliferation of T-cellsand vascular smooth muscle cells.

When referring to antibody, it is to include intact monoclonalantibodies, nanobodies, polyclonal antibodies, multi-specific antibodiesformed from at least 2 intact antibodies, and antibodies fragments solong as they exhibit the desired biological activity.

The phrase “compound which targets, decreases or inhibits the activityof a protein or lipid phosphatase” as used herein includes but is notlimited to inhibitors of phosphatase 1, phosphatase 2A, PTEN or CDC25,e.g., okadaic acid or a derivative thereof

The term “monoclonal antibodies”, as used herein, includes, but is notlimited to bevacizumab, cetuximab, trastuzumab, Ibritumomab tiuxetan,denosumab, anti-CD40, anti-GM-CSF, and tositumomab and iodine I 131.Bevacizumab can be administered in the form as it is marketed, e.g.AVASTIN; cetuximab as ERBITUX; trastuzumab as HERCEPTIN; Rituximab asMABTHERA; Ibritumomab tiuxetan as ZEVULIN; anti-RANKL as denosumab (AMG162), anti-CD40 as HCD122 (U.S. patent application 2002-0106371), andtositumomab and iodine I 131 as BEXXAR.

The phrase “further anti-angiogenic compounds” includes but is notlimited to compounds having another mechanism for their activity, e.g.,unrelated to protein or lipid kinase inhibition, e.g., thalidomide(THALOMID) and TNP-470.

The phrase “compounds which induce cell differentiation processes” asused herein, include but is not limited to retinoic acid, α-, γ- orδ-tocopherol or α-, γ- or δ-tocotrienol.

The term “cyclooxygenase inhibitor” as used herein includes, but is notlimited to, e.g., Cox-2 inhibitors, 5-alkyl substituted2-arylaminophenylacetic acid and derivatives, such as celecoxib(CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a5-alkyl-2-arylaminophenylacetic acid, e.g.,5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid, lumiracoxib.

The term “bisphosphonates”, as used herein, includes, but is not limitedto, etridonic, clodronic, tiludronic, pamidronic, alendronic,ibandronic, risedronic and zoledronic acid. “Etridonic acid” can beadministered, e.g., in the form as it is marketed, e.g., DIDRONEL;“clodronic acid” as BONEFOS; “tiludronic acid” as SKELID; “pamidronicacid” as AREDIA; “alendronic acid” as FOSAMAX; “ibandronic acid” asBONDRANAT; “risedronic acid” as ACTONEL; and “zoledronic acid” asZOMETA.

The term “heparanase inhibitor”, as used herein, refers to compoundswhich target, decrease or inhibit heparin sulphate degradation. The termincludes, but is not limited to, PI 88.

The term “biological response modifier”, as used herein, includes, butis not limited to lymphokine or interferons, e.g., interferon γ.

The term “inhibitor of Ras oncogenic isoforms”, as used herein,includes, but is not limited to H-Ras, K-Ras or N-Ras, as used herein,refers to compounds which target, decrease or inhibit the oncogenicactivity of Ras, e.g., a farnesyl transferase inhibitor (FTI), e.g.,L-744832, DK8G557 or R115777 (ZARNESTRA).

The term “telomerase inhibitor”, as used herein, includes, but is notlimited to compounds which target, decrease or inhibit the activity oftelomerase. Compounds which target, decrease or inhibit the activity oftelomerase are especially compounds which inhibit the telomerasereceptor, e.g., telomestatin.

The term “matrix metalloproteinase inhibitor” or (MMP inhibitor), asused herein, includes, but is not limited to, collagen peptidomimeticand non-peptidomimetic inhibitors; tetracycline derivatives, e.g.,hydroxamate peptidomimetic inhibitor batimastat; and itsorally-bioavailable analogue marimastat (BB-2516), prinomastat (AG3340),metastat (NSC 683551) BMS 279251, BAY 12-9566, TAA211, MMI270B orAAJ996.

The term “methionine aminopeptidase inhibitor”, as used herein,includes, but is not limited to, compounds which target, decrease orinhibit the activity of methionine aminopeptidase. Compounds whichtarget, decrease or inhibit the activity of methionine aminopeptidaseare, e.g., bengamide or a derivative thereof.

The term “proteasome inhibitors”, as used herein, includes compoundswhich target, decrease or inhibit the activity of the proteosome.Compounds which target, decrease or inhibit the activity of theproteosome include, but are not limited to, PS-341; MLN 341. bortezomibor Velcade.

The phrase “agent used in the treatment of hematologic malignancies”, asused herein, includes, but is not limited to, FMS-like tyrosine kinaseinhibitors, e.g., compounds targeting, decreasing or inhibiting theactivity of FMS-like tyrosine kinase receptors (Flt-3R); interferon,1-b-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors,e.g., compounds which target, decrease or inhibit anaplastic lymphomakinase.

The phrase “compounds which target, decrease or inhibit the activity ofFlt-3” as used herein, includes, but is not limited to compounds,proteins or antibodies which inhibit Flt-3, e.g.,N-benzoyl-staurosporine, midostaurin, a staurosporine derivative,SU11248 and MLN518.

The term “HSP90 inhibitors”, as used herein, includes, but is notlimited to, compounds targeting, decreasing or inhibiting the intrinsicATPase activity of HSP90; degrading, targeting, decreasing or inhibitingthe HSP90 client proteins via the ubiquitin proteosome pathway.Compounds targeting, decreasing or inhibiting the intrinsic ATPaseactivity of HSP90 are especially compounds, proteins or antibodies whichinhibit the ATPase activity of HSP90, e.g.,17-allylamino,17-demethoxygeldanamycin (17AAG), a geldanamycinderivative; other geldanamycin-related compounds; radicicol and HDACinhibitors.

The term “an antiproliferative antibody” as used herein, includes, butis not limited to trastuzumab (HERCEPTIN), trastuzumab-DM1, erlotinib(TARCEVA), bevacizumab (AVASTIN), rituximab (RITUXAN), PRO64553(anti-CD40) and 2C4 Antibody. By antibodies is meant e.g. intactmonoclonal antibodies, polyclonal antibodies, multispecific antibodiesformed from at least 2 intact antibodies, and antibodies fragments solong as they exhibit the desired biological activity.

The term “HDAC inhibitor”, as used herein relates to relates tocompounds which inhibit the histone deacetylase and which possessanti-proliferative activity. This includes but is not limited tocompounds disclosed in WO 02/22577, especiallyN-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide,andN-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamideand pharmaceutically acceptable salts thereof (LBH589). It furtherespecially includes Suberoylanilide hydroxamic acid (SAHA);[4-(2-amino-phenylcarbamoyl)-benzyl]-carbamic acid pyridine-3-ylmethylester and derivatives thereof; butyric acid, pyroxamide, trichostatin A,Oxamflatin, apicidin, Depsipeptide; depudecin and trapoxin.

The phrase “compound which targets, decreases or inhibits theactivity/function of serine/theronine mTOR kinase” as used herein,includes but is not limited to compounds, proteins or antibodies whichtarget/inhibit members of the mTOR kinase family, e.g., RAD, RAD001,CCI-779, ABT578, SAR543, rapamycin and derivatives/analogs thereof,AP23573 and AP23841 from Ariad, everolimus (CERTICAN) and sirolimus(RAPAMUNE), CCI-779 and ABT578. CERTICAN (everolimus, RAD) aninvestigational novel proliferation signal inhibitor that preventsproliferation of T-cells and vascular smooth muscle cells.

The term “somatostatin receptor antagonist”, as used herein, includes,but is not limited to, agents which target, treat or inhibit thesomatostatin receptor, such as octreoride and SOM230.

The term “anti-leukemic compound” as used herein, includes, but is notlimited to Ara-C, a pyrimidine analog, which is the 2′-α-hydroxy ribose(arabinoside) derivative of deoxycytidine. Also included is the purineanalog of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabinephosphate.

The phrase “tumor cell damaging approaches” refers to approaches, suchas ionizing radiation. The term “ionizing radiation”, referred to aboveand hereinafter, means ionizing radiation that occurs as eitherelectromagnetic rays, such as X-rays and gamma rays; or particles, suchas alpha, beta and gamma particles. Ionizing radiation is provided in,but not limited to, radiation therapy and is known in the art. SeeHellman, Cancer, 4th Edition, Vol. 1, Devita et al., Eds., pp. 248-275(1993).

The term “EDG binder” as used herein, includes, but is not limited to, aclass of immunosuppressants that modulates lymphocyte recirculation,such as FTY720.

The term “ribonucleotide reductase inhibitor” as used herein, includes,but is not limited to, pyrimidine or purine nucleoside analogsincluding, but not limited to, fludarabine and/or ara-C; 6-thioguanine;5-FU; cladribine; 6-mercaptopurine, especially in combination with ara-Cagainst ALL; and/or pentostatin. Ribonucleotide reductase inhibitors areespecially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives,such as PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL 8. See Nandy etal., Acta Oncologica, Vol. 33, No. 8, pp. 953-961 (1994).

The term “S-adenosylmethionine decarboxylase inhibitors”, as usedherein, includes, but is not limited to, the compounds disclosed in U.S.Pat. No. 5,461,076.

The phrase “monoclonal antibodies of VEGF or VEGFR”, as used herein,includes but is not limited to, compounds disclosed in WO 98/35958,e.g., 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or apharmaceutically acceptable salt thereof, e.g., the succinate, or in WO00/09495, WO 00/27820, WO 00/59509, WO 98/11223, WO 00/27819 and EP 0769 947; those as described by Prewett et al., Cancer Res, Vol. 59, pp.5209-5218 (1999); Yuan et al., Proc Natl Acad Sci USA, Vol. 93, pp.14765-14770 (1996); Zhu et al., Cancer Res, Vol. 58, pp. 3209-3214(1998); and Mordenti et al., Toxicol Pathol, Vol. 27, No. 1, pp. 14-21(1999) in WO 00/37502 and WO 94/10202; ANGIOSTATIN, described byO'Reilly et al., Cell, Vol. 79, pp. 315-328 (1994); ENDOSTATIN,described by O'Reilly et al., Cell, Vol. 88, pp. 277-285 (1997);anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; oranti-VEGF antibodies or anti-VEGF receptor antibodies, e.g., rhuMAb andRHUFab; VEGF aptamer, e.g., Macugon; FLT-4 inhibitors; FLT-3 inhibitors;VEGFR-2 IgG1 antibody; Angiozyme (RPI 4610); and Avastan.

The term “photodynamic therapy”, as used herein, refers to therapy whichuses certain chemicals known as photosensitizing agents to treat orprevent cancers. Examples of photodynamic therapy include, but are notlimited to, treatment with agents, such as, e.g., VISUDYNE and porfimersodium.

The term “angiostatic steroid”, as used herein, includes, but is notlimited to agents which block or inhibit angiogenesis, such as, e.g.,anecortave, triamcinolone, hydrocortisone, 11-α-epihydrocotisol,cortexolone, 17α-hydroxyprogesterone, corticosterone,desoxycorticosterone, testosterone, estrone and dexamethasone.

The phrase “Implant containing corticosteroids” as used herein,includes, but is not limited to agents, such as, e.g., fluocinolone anddexamethasone.

The term “AT1 receptor antagonist” as used herein, includes, but is notlimited to agents, such as DIOVAN.

The term “ACE inhibitor” as used herein, includes, but is not limited toCIBACEN, benazepril, enazepril (LOTENSIN), captopril, enalapril,fosinopril, lisinopril, moexipril, quinapril, ramipril, perindopril andtrandolapril.

Other pharmaceutically active agents include, but are not limited to,plant alkaloids, hormonal agents and antagonists, biological responsemodifiers, preferably lymphokines or interferons, antisenseoligonucleotides or oligonucleotide derivatives; or miscellaneous agentsor agents with other or unknown mechanism of action.

In each case where citations of patent applications or scientificpublications are given, in particular with regard to the respectivecompound claims and the final products of the working examples therein,the subject matter of the final products, the pharmaceuticalpreparations and the claims is hereby incorporated into the presentapplication by reference to these publications. Comprised are likewisethe corresponding stereoisomers, as well as the corresponding crystalmodifications, e.g., solvates and polymorphs, which are disclosedtherein. The compounds used as active ingredients in the combinationsdisclosed herein can be prepared and administered as described in thecited documents, respectively.

The structure of the active agents identified by code numbers, genericor trade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g., PatentsInternational, e.g., IMS World Publications, or the publicationsmentioned above and below. The corresponding content thereof is herebyincorporated by reference.

It will be understood that references to the components (a) and (b) aremeant to also include the pharmaceutically acceptable salts of any ofthe active substances. If active substances comprised by components (a)and/or (b) have, for example, at least one basic center, they can formacid addition salts. Corresponding acid addition salts can also beformed having, if desired, an additionally present basic center. Activesubstances having an acid group, e.g., COOH, can form salts with bases.The active substances comprised in components (a) and/or (b) or apharmaceutically acceptable salts thereof may also be used in form of ahydrate or include other solvents used for crystallization.

Thus, in a first aspect, the present invention relates to a method forthe prevention of treatment of proliferative diseases or diseases thatare triggered by persistent angiogenesis in a mammal, preferably a humanpatient, which comprises treating the patient concurrently orsequentially with pharmaceutically effective amounts of a combinationof:

(a) a neutralizing anti-DKK1/4 composition; and

(b) an pharmaceutically active agent.

In preferred embodiment, the present invention provides a pharmaceuticalpreparation comprising:

(a) a neutralizing anti-DKK1/4 composition; and

(b) one or more pharmaceutically active agents selected from the groupconsisting of an aromatase inhibitor; an antiestrogen; an anti-androgen;a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase IIinhibitor; a microtubule active agent; an alkylating agent; ananti-neoplastic anti-metabolite; a platin compound; a compoundtargeting/decreasing a protein or lipid kinase activity or a protein orlipid phosphatase activity, a anti-angiogenic compound; a compound whichinduces cell differentiation processes; monoclonal antibodies; acyclooxygenase inhibitor; a bisphosphonate; a heparanase inhibitor; abiological response modifier; an inhibitor of Ras oncogenic isoforms; atelomerase inhibitor; a protease inhibitor, a matrix metalloproteinaseinhibitor, a methionine aminopeptidase inhibitor; a proteasomeinhibitor; agents which target, decrease or inhibit the activity ofFlt-3; an HSP90 inhibitor; antiproliferative antibodies; an HDACinhibitor; a compound which targets, decreases or inhibits theactivity/function of serine/theronine mTOR kinase; a somatostatinreceptor antagonist; an anti-leukemic compound; tumor cell damagingapproaches; an EDG binder; a ribonucleotide reductase inhibitor; anS-adenosylmethionine decarboxylase inhibitor; a monoclonal antibody ofVEGF or VEGFR; photodynamic therapy; an Angiostatic steroid; an implantcontaining corticosteroids; an AT1 receptor antagonist; and an ACEinhibitor.

Any of the combination of components (a) and (b), the method of treatinga warm-blooded animal comprising administering these two components, apharmaceutical composition comprising these two components forsimultaneous, separate or sequential use, the use of the combination forthe delay of progression or the treatment of a proliferative disease orfor the manufacture of a pharmaceutical preparation for these purposesor a commercial product comprising such a combination of components (a)and (b), all as mentioned or defined above, will be referred tosubsequently also as combination of the invention (so that this termrefers to each of these embodiments which thus can replace this termwhere appropriate).

Simultaneous administration may, e.g., take place in the form of onefixed combination with two or more active ingredients, or bysimultaneously administering two or more active ingredients that areformulated independently. Sequential use (administration) preferablymeans administration of one (or more) components of a combination at onetime point, other components at a different time point, that is, in achronically staggered manner, preferably such that the combination showsmore efficiency than the single compounds administered independently(especially showing synergism). Separate use (administration) preferablymeans administration of the components of the combination independentlyof each other at different time points, preferably meaning that thecomponents (a) and (b) are administered such that no overlap ofmeasurable blood levels of both compounds are present in an overlappingmanner (at the same time).

Also combinations of two or more of sequential, separate andsimultaneous administration are possible, preferably such that thecombination component-drugs show a joint therapeutic effect that exceedsthe effect found when the combination component-drugs are usedindependently at time intervals so large that no mutual effect on theirtherapeutic efficiency can be found, a synergistic effect beingespecially preferred.

The term “delay of progression” as used herein means administration ofthe combination to patients being in a pre-stage or in an early phase,of the first manifestation or a relapse of the disease to be treated, inwhich patients, e.g., a pre-form of the corresponding disease isdiagnosed or which patients are in a condition, e.g., during a medicaltreatment or a condition resulting from an accident, under which it islikely that a corresponding disease will develop.

“Jointly therapeutically active” or “joint therapeutic effect” meansthat the compounds may be given separately (in a chronically staggeredmanner, especially a sequence-specific manner) in such time intervalsthat they preferably, in the warm-blooded animal, especially human, tobe treated, still show a (preferably synergistic) interaction (jointtherapeutic effect). Whether this is the case, can inter alia bedetermined by following the blood levels, showing that both compoundsare present in the blood of the human to be treated at least duringcertain time intervals.

“Pharmaceutically effective” preferably relates to an amount that istherapeutically or in a broader sense also prophylactically effectiveagainst the progression of a proliferative disease.

The term “a commercial package” or “a product”, as used herein definesespecially a “kit of parts” in the sense that the components (a) and (b)as defined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the components (a) and (b),i.e., simultaneously or at different time points. Moreover, these termscomprise a commercial package comprising (especially combining) asactive ingredients components (a) and (b), together with instructionsfor simultaneous, sequential (chronically staggered, in time-specificsequence, preferentially) or (less preferably) separate use thereof inthe delay of progression or treatment of a proliferative disease. Theparts of the kit of parts can then, e.g., be administered simultaneouslyor chronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts. Verypreferably, the time intervals are chosen such that the effect on thetreated disease in the combined use of the parts is larger than theeffect which would be obtained by use of only any one of the combinationpartners (a) and (b) (as can be determined according to standardmethods. The ratio of the total amounts of the combination partner (a)to the combination partner (b) to be administered in the combinedpreparation can be varied, e.g., in order to cope with the needs of apatient sub-population to be treated or the needs of the single patientwhich different needs can be due to the particular disease, age, sex,body weight, etc. of the patients. Preferably, there is at least onebeneficial effect, e.g., a mutual enhancing of the effect of thecombination partners (a) and (b), in particular a more than additiveeffect, which hence could be achieved with lower doses of each of thecombined drugs, respectively, than tolerable in the case of treatmentwith the individual drugs only without combination, producing additionaladvantageous effects, e.g., less side effects or a combined therapeuticeffect in a non-effective dosage of one or both of the combinationpartners (components) (a) and (b), and very preferably a strongsynergism of the combination partners (a) and (b).

Both in the case of the use of the combination of components (a) and (b)and of the commercial package, any combination of simultaneous,sequential and separate use is also possible, meaning that thecomponents (a) and (b) may be administered at one time pointsimultaneously, followed by administration of only one component withlower host toxicity either chronically, e.g., more than 3-4 weeks ofdaily dosing, at a later time point and subsequently the other componentor the combination of both components at a still later time point (insubsequent drug combination treatment courses for an optimal anti-tumoreffect) or the like.

The COMBINATION OF THE INVENTION can also be applied in combination withother treatments, e.g., surgical intervention, hyperthermia and/orirradiation therapy.

The pharmaceutical compositions according to the present invention canbe prepared by conventional means and are those suitable for enteral,such as oral or rectal, and parenteral administration to mammalsincluding man, comprising a therapeutically effective amount of a VEGFinhibitor and at least one pharmaceutically active agent alone or incombination with one or more pharmaceutically acceptable carriers,especially those suitable for enteral or parenteral application.

The pharmaceutical compositions comprise from about 0.00002 to about100%, especially, e.g., in the case of infusion dilutions that are readyfor use, of 0.0001 to 0.02%, or, e.g., in case of injection or infusionconcentrates or especially parenteral formulations, from about 0.1% toabout 95%, preferably from about 1% to about 90%, more preferably fromabout 20% to about 60% active ingredient (weight by weight, in eachcase). Pharmaceutical compositions according to the invention may be,e.g., in unit dose form, such as in the form of ampoules, vials,dragées, tablets, infusion bags or capsules.

The effective dosage of each of the combination partners employed in aformulation of the present invention may vary depending on theparticular compound or pharmaceutical compositions employed, the mode ofadministration, the condition being treated and the severity of thecondition being treated. A physician, clinician or veterinarian ofordinary skill can readily determine the effective amount of each of theactive ingredients necessary to prevent, treat or inhibit the progressof the condition.

Tyrphostins, especially Adaphostin, are preferably administered to awarm-blooded animal, especially a human in a dosage in the range ofabout 1-6000 mg/day, more preferably 25-5000 mg/day, most preferably50-4000 mg/day. Unless stated otherwise herein, the compound ispreferably administered from one to 5, especially from 1-4 times perday.

Pharmaceutical preparations for the combination therapy for enteral orparenteral administration are, e.g., those in unit dosage forms, such assugar-coated tablets, capsules or suppositories, and furthermoreampoules. If not indicated otherwise, these formulations are prepared byconventional means, e.g., by means of conventional mixing, granulating,sugar-coating, dissolving or lyophilizing processes. It will beappreciated that the unit content of a combination partner contained inan individual dose of each dosage form need not in itself constitute aneffective amount since the necessary effective amount can be reached byadministration of a plurality of dosage units. One of skill in the arthas the ability to determine appropriate pharmaceutically effectiveamounts of the combination components.

Preferably, the compounds or the pharmaceutically acceptable saltsthereof, are administered as an oral pharmaceutical formulation in theform of a tablet, capsule or syrup; or as parenteral injections ifappropriate.

In preparing compositions for oral administration, any pharmaceuticallyacceptable media may be employed such as water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents. Pharmaceuticallyacceptable carriers include starches, sugars, microcrystallinecelluloses, diluents, granulating agents, lubricants, binders,disintegrating agents.

Solutions of the active ingredient, and also suspensions, and especiallyisotonic aqueous solutions or suspensions, are useful for parenteraladministration of the active ingredient, it being possible, e.g., in thecase of lyophilized compositions that comprise the active ingredientalone or together with a pharmaceutically acceptable carrier, e.g.,mannitol, for such solutions or suspensions to be produced prior to use.The pharmaceutical compositions may be sterilized and/or may compriseexcipients, e.g., preservatives, stabilizers, wetting and/or emulsifyingagents, solubilizers, salts for regulating the osmotic pressure and/orbuffers, and are prepared in a manner known per se, e.g., by means ofconventional dissolving or lyophilizing processes. The solutions orsuspensions may comprise viscosity-increasing substances, such as sodiumcarboxymethylcellulose, carboxymethylcellulose, dextran,polyvinylpyrrolidone or gelatin. Suspensions in oil comprise as the oilcomponent the vegetable, synthetic or semi-synthetic oils customary forinjection purposes.

The isotonic agent may be selected from any of those known in the art,e.g. mannitol, dextrose, glucose and sodium chloride. The infusionformulation may be diluted with the aqueous medium. The amount ofaqueous medium employed as a diluent is chosen according to the desiredconcentration of active ingredient in the infusion solution. Infusionsolutions may contain other excipients commonly employed in formulationsto be administered intravenously such as antioxidants.

The present invention further relates to “a combined preparation”,which, as used herein, defines especially a “kit of parts” in the sensethat the combination partners (a) and (b) as defined above can be dosedindependently or by use of different fixed combinations withdistinguished amounts of the combination partners (a) and (b), i.e.,simultaneously or at different time points. The parts of the kit ofparts can then, e.g., be administered simultaneously or chronologicallystaggered, that is at different time points and with equal or differenttime intervals for any part of the kit of parts. The ratio of the totalamounts of the combination partner (a) to the combination partner (b) tobe administered in the combined preparation can be varied, e.g., inorder to cope with the needs of a patient sub-population to be treatedor the needs of the single patient based on the severity of any sideeffects that the patient experiences.

Uses and Methods of the Invention

The antibodies (and immunoconjugates and bispecific molecules) of thepresent invention have in vitro and in vivo diagnostic and therapeuticutilities. For example, these molecules can be administered to cells inculture, e.g. in vitro or in vivo, or in a subject, e.g., in vivo, totreat, prevent or diagnose a variety of disorders. The term “subject” asused herein in intended to include human and non-human animals.Non-human animals includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles. The methods are particularlysuitable for treating human patients having a disorder associated withDKK1 expression. When antibodies to DKK1 are administered together withanother agent, the two can be administered in either order orsimultaneously.

In one embodiment, the antibodies (and immunoconjugates and bispecificmolecules) of the invention can be used to detect levels of DKK1, orlevels of cells that contain DKK1. This can be achieved, for example, bycontacting a sample (such as an in vitro sample) and a control samplewith the anti-DKK1 antibody under conditions that allow for theformation of a complex between the antibody and DKK1. Any complexesformed between the antibody and DKK1 are detected and compared in thesample and the control. By non-limiting example, standard detectionmethods that are well known in the art, such as e.g., ELISA, MALDI andflow cytometic assays, can be performed using the compositions of theinvention.

Accordingly, in one aspect, the invention further provides methods fordetecting the presence of DKK1 (e.g., human DKK1 antigen) in a sample,or measuring the amount of DKK1, comprising contacting the sample, and acontrol sample, with an antibody of the invention, or an antigen bindingportion thereof, which specifically binds to DKK1, under conditions thatallow for formation of a complex between the antibody or portion thereofand DKK1. The formation of a complex is then detected, wherein adifference in complex formation between the sample compared to thecontrol sample is indicative of the presence of DKK1 in the sample.

Also within the scope of the invention are kits consisting of thecompositions (e.g., antibodies, human antibodies, immunoconjugates andbispecific molecules) of the invention and instructions for use. The kitcan further contain a least one additional reagent, or one or moreadditional antibodies of the invention (e.g., an antibody having acomplementary activity which binds to an epitope on the target antigendistinct from the first antibody). Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

The invention having been fully described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references, including issued patents and publishedpatent applications, cited throughout this application are herebyincorporated by reference.

EXAMPLES Example 1 Generation of Human DKK1-Specific Antibodies from theHuCAL GOLD® Library

Therapeutic antibodies against human DKK1 protein are generated byselection of clones having high binding affinities, using as the sourceof antibody variant proteins a commercially available phage displaylibrary, the MorphoSys HuCAL GOLD® library. HuCAL GOLD® is a Fab library(Knappik et al., 2000 J. Mol. Biol. 296:57-86; Krebs et al., 2001 JImmunol. Methods 254:67-84; Rauchenberger et al., 2003 J Biol Chem.278(40):38194-38205), in which all six CDRs are diversified byappropriate mutation, and which employs the CysDisplay™ technology forlinking Fab fragments to the phage surface (WO 01/05950 Löhning 2001).

General Procedures: Phagemid Rescue, Phage Amplification, andPurification

The HuCAL GOLD® library is amplified in standard rich bacterial medium(2×YT) containing 34 μg/ml chloramphenicol and 1% glucose (2×YT-CG).After infection of cells at an OD_(600nm) of 0.5 with VCSM13 helperphages (incubating the mix of cells and phage for 30 min at 37° C.without shaking followed by 30 min at 37° C. shaking at 250 rpm), cellsare centrifuged (4120 g; 5 min; 4° C.), are resuspended in 2×YT/34 μg/mlchloramphenicol/50 μg/ml kanamycin/0.25 mM IPTG, and are grown overnightat 22° C. At the end of this period cells are removed by centrifugation,and phages are PEG-precipitated twice from the supernatant, resuspendedin PBS/20% glycerol and are stored at −a 80° C.

Phage amplification between two panning rounds is conducted as follows:mid-log phase E. coli strain TG1 cells are infected with phages areeluted following the selection with DKK1 protein, and are plated ontoLB-agar supplemented with 1% of glucose and 34 μg/ml of chloramphenicol(LB-CG plates). After overnight incubation of the plates at 30° C.,bacterial colonies are scraped off the agar surface, and used toinoculate 2×YT-CG broth to obtain an OD_(600nm) of 0.5, then VCSM13helper phages are added to obtain a productive infection as describedabove.

Pre-Experiments for Solution Panning Using Strep-Tactin Magnetic Beads

The Strep-tag II has been reported to have low affinity for theStrep-Tactin matrix (K_(D)˜1 μM according to (Voss and Skerra, 1997Protein Eng. 10:975-982), therefore, a pre-experiment is performed toassess the suitability of using Strep-Tactin-coated MagStrep beads forthe capturing of the antigen during the antibody selections, and toavoid antigen loss during the pannings.

For that purpose, 8 mg of MagStrep beads is incubated with 46 μg ofHis-Strep-tagged DKK1 for 1 h at room temperature and the sample isdivided into four pre-blocked Eppendorf tubes. One tube served as thepositive control (no washing) and the other three samples are washedwith different stringencies according to the HuCAL GOLD® manual panningsection. Detection of binding of the His-Strep-tagged DKK1 to theMagStrep beads (Strep-Tactin coated Magnetic beads obtained from IBA,Göttingen, Germany) is performed in BioVeris using a goat anti-DKK1antibody and a Rubidium-labeled anti-goat detection antibody.

As shown in FIG. 1 herein, no significant loss of His-Strep-tagged DKK1from the Strep-Tactin-coated beads is detectable when the non-washedbeads are compared with those beads washed with different HuCAL®stringencies. Thus, the His-Strep-tagged DKK1 seemed to be suitable forthe use in the solution pannings with Strep-Tactin-coated magnetic beads(MagStrep beads).

Selection by Panning of DKK1-Specific Antibodies from the Library

For the selection of antibodies recognizing human DKK1, two panningstrategies are applied.

In summary, HuCAL GOLD® phage-antibodies are divided into four poolscomprising different combinations of V_(H) master genes (pool 1contained VH1/5λκ; pool 2 contained V_(H)3λκ; pool 3 containedV_(H)2/4/6λκ; and pool 4 contained V_(H)1-6λκ). These pools areindividually subjected to two rounds of solution panningHis-Strep-tagged DKK1 captured onto StrepTactin magnetic beads (MegaStrep beads; IBA), and for the third selection round only, either onHis-Strep-tagged DKK1 captured onto StrepTactin magnetic beads or onAPP-tagged human DKK1 protein captured by Streptavidin beads (Dynabeads®M-280 Streptavidin; Dynal) with a biotinylated anti-APP antibody.

In detail, for the solution panning using His-Strep-tagged DKK1 coupledto StrepTactin magnetic beads, the following protocol is applied:pre-blocked tubes are prepared (1.5 ml Eppendorf tubes) by treatmentwith 1.5 ml 2× ChemiBLOCKER diluted 1:1 with PBS over night at 4° C.Pre-blocked beads are prepared by treatment as follows: 580 μl (28 mgbeads) StrepTactin magnetic beads are washed once with 580 μl PBS andresuspended in 580 μl 1× ChemiBLOCKER (diluted in one volume 1×PBS).Blocking of the beads is performed in the pre-blocked tubes over nightat 4° C.

Phage particles diluted in PBS to a final volume of 500 μl for eachpanning condition are mixed with 500 μl 2× ChemiBLOCKER/0.1% Tween andkept for one hour at room temperature on a rotating wheel.Pre-adsorption of phage particles for removal of StrepTactin orbeads-binding phages is performed twice: 160 μl of blocked StrepTactinmagnetic beads (4 mg) is added to the blocked phage particles, and isincubated for 30 min at room temperature on a rotating wheel. Afterseparation of the beads by a magnetic device (Dynal MPC-E), the phagesupernatant (˜1.1 ml) is transferred to a fresh, blocked reaction tubeand pre-adsorption is repeated using 160 μl blocked beads for 30 min.Then, His-Strep-tagged DKK1, either 400 nM or 100 nM, is added to theblocked phage particles in a fresh, blocked 1.5 ml reaction tube and themixture is incubated for 60 min at room temperature on a rotating wheel.

The phage-antigen complexes are captured using either 320 μl or 160 μlof blocked StrepTactin magnetic beads added to the 400 nM or the 100 nMphage panning pools, respectively, which is then incubated for 20 min atroom temperature on a rotating wheel. Phage particles bound to theStrepTactin magnetic beads are again collected with the magneticparticle separator.

Beads are then washed seven times with PBS/0.05% Tween (PBST), followedby washing another three times with PBS only. Elution of phage particlesfrom the StrepTactin magnetic beads is performed by addition of 200 μl20 mM DTT in 10 mM Tris-HCl, pH 8.0 to each tube for 10 min. The eluateis collected, and the beads are washed once with 200 μl PBS and the PBSeluate is added to the DTT eluate. This eluate sample is used to infect14 ml of an E. coli TG-1 culture that had been grown to an OD_(600nm) of0.6-0.8.

After infection and subsequent centrifugation for 10 min at 5000 rpm,each bacterial pellet is resuspended in 500 μl 2×YT medium, plated onto2×YT-CG agar plates and incubated overnight at 30° C. The next morning,the resulting colonies are scraped off the plates and the phage isprepared by rescue and amplification as described above.

The second round of solution pannings on His-Strep-tagged DKK1 isperformed according to the protocol of the first round, except thatdecreasing amounts of antigen are used (50 nM, and 10 nM) and thestringency of the washing procedure is altered appropriately.

Two different panning strategies are applied for the third selectionround: the amplified phage output of the second panning round is splitand subjected to two different panning conditions. The first half of thephage output is used for the standard panning strategy on humanHis-Strep-tagged DKK1 captured onto StrepTactin beads as described above(antigen amounts are 10 nM or 1 nM, respectively).

The second panning variation for the third selection round is performedon human APP-tagged DKK1. APP-tagged DKK1 protein at a finalconcentration of 50 nM or 10 nM is mixed with 1 ml of pre-cleared,second round phage particles, and the mixture is incubated at roomtemperature for 1 hour on a rotating wheel. In parallel, 8 mgpre-blocked Dynabeads M-280 Streptavidin (Dynal) is incubated with 40 μgbiotinylated mouse anti-APP antibody for 30 min at room temperature on arotating wheel followed by two washing steps with PB ST. The pre-formedcomplexes consisting of phage-antibodies bound to APP-tagged DKK1 arecaptured by the anti-APP coated M-280 Streptavidin magnetic beads for 30min at room temperature. Phage elution and amplification are performedas described above.

Subcloning and Expression of Soluble Fab Fragments

The Fab-encoding inserts of the selected HuCAL GOLD® phagemids aresubcloned into expression vector pMORPH®X9_Fab_FH, in order tofacilitate rapid and efficient expression of soluble Fabs. For thispurpose, the plasmid DNA of the selected clones is digested withrestriction enzyme endonucleases XbaI and EcoRI, thereby excising theFab-encoding insert (ompA-VLCL and phoA-Fd). This insert is then clonedinto XbaI/EcoRI-digested expression vector pMORPH®X9_Fab_FH.

Fab proteins are expressed from this vector, and as a result carry twoC-terminal tags (FLAG™ and 6×His, respectively) for both detection andpurification.

Microexpression of HuCAL GOLD® Fab Antibodies in E. coli

To obtain sufficient amounts of protein encoded by each of the clonesobtained above, chloramphenicol-resistant single bacterial colonies areselected after subcloning of the selected Fabs into the pMORPH®X9_Fab_FHexpression vector. Each of these colonies is then used to inoculate thewells of a sterile 96-well microtiter plate, each well containing 100 μl2×YT-CG medium per well, and bacteria are grown overnight at 37° C. Asample (5 μl) of each E. coli TG-1 culture is transferred to a fresh,sterile 96-well microtiter plate pre-filled with 100 μl 2×YT mediumsupplemented with 34 μg/ml chloramphenicol and 0.1% glucose per well.The microtiter plates are incubated at 30° C. with shaking at 400 rpm ona microplate shaker until the cultures are slightly turbid (˜2-4 hrs)with an OD_(600nm) of about 0.5.

For expression in the format of these plates, 20 μl 2×YT mediumsupplemented with 34 μg/ml chloramphenicol and 3 mM IPTG(isopropyl-β-D-thiogalactopyranoside) is added per well (finalconcentration 0.5 mM IPTG), the microtiter plates sealed with agas-permeable tape, and incubated overnight at 30° C. shaking at 400rpm.

Generation of Whole Cell Lysates (BEL Extracts)

To each well of the expression plates, 40 μl BEL buffer (2×BBS/EDTA:24.7 g/1 boric acid, 18.7 g NaCl/1, 1.49 g EDTA/1, pH 8.0) containing2.5 mg/ml lysozyme is added, and plates are incubated for 1 h at 22° C.on a microtiter plate shaker (400 rpm). The BEL extracts are used forbinding analysis by FMAT (see Example 2).

Expression of Microgram Amounts of HuCAL GOLD® Fab Antibodies in E. coliand Purification

Expression of Fab fragments encoded by pMORPH®X9_Fab_FH in E. coli TG1F-cells is carried out in 50 ml plastic tubes. For this purpose,pre-cultures inoculated with single clones are grown in 2×YT-CG mediumovernight at 30° C. The next morning, 50 μl of each pre-culture are usedto inoculate 25 ml 2×YT medium supplemented with 34 μg/mlChloramphenicol, 1 mM IPTG, and 0.1% glucose in sterile 50 ml plastictubes, and incubated over night at 30° C. E. coli cells are harvested,the cell pellets frozen and finally disrupted with Bug Buster (Novagen).The Fab fragments are isolated using Ni-NTA Agarose (Qiagen, Hilden,Germany).

Expression of Milligram Amounts of HuCAL GOLD Fab Antibodies in E. coliand Purification

Expression of Fab fragments encoded by pMORPH®X9_Fab_FH in TG1 F-cellsis carried out in shaker flask cultures using 750 ml of 2×YT mediumsupplemented with 34 μg/ml chloramphenicol. Cultures are shaken at 30°C. until the OD_(600nm) reached 0.5. Expression is induced by additionof 0.75 mM IPTG followed by incubation for 20 h at 30° C. Cells aredisrupted using lysozyme, and Fab fragments are isolated by Ni-NTAchromatography (Qiagen, Hilden, Germany). Protein concentrations aredetermined by UV-spectrophotometry (Krebs et al., 2001).

Example 2 Identification of DKK1-Specific HuCAL® Antibodies

BEL extracts of individual E. coli clones selected by the abovementioned panning strategies are analyzed by Fluorometric MicrovolumeAssay Technology (FMAT™, 8200 Cellular Detection System analyzer,Applied Biosystems, Foster City, Calif.), to identify clones encodingDKK1-specific Fabs. The FMAT™ 8100 HTS System is a fluorescencemacro-confocal, high-throughput screening instrument that automatesdetection of mix-and-read, non-radioactive assays with live cells orbeads (Miraglia, J. Biomol. Screening (1999), 4(4) 193-204).

Fluorometric Microvolume Assay Technology-Based Binding Analysis (FMAT)for Detection of DKK1-Binding Fabs from Bacterial Lysates

For the detection of DKK1-binding Fab antibodies from E. coli lysates(BEL extracts), binding is analyzed with the FMAT 8200 cellulardetection system (Applied Biosystems). To couple His-Strep-tagged DKK1onto M-450 Epoxy beads (Dynal), a sample of 300 μl M-450 Epoxy beads(1.2×10⁸ beads) is transferred into a reaction tube and captured with amagnetic particle separator. The supernatant is removed and the beadsare washed four times in 1 ml of 100 mM sodium phosphate buffer, pH 7.4.For antigen coating, 60 μg His-Strep-tagged DKK1 is added to the beadsuspension in 150 μl 100 mM sodium phosphate buffer, pH 7.4. Theantigen-bead suspension is incubated for 16 h at room temperature on arotating wheel. The coated beads are then washed three times with PBSand resuspended in a final volume of 250 μl PBS.

For each 384-well plate, a mixture of 20 ml PBS containing 3% BSA,0.005% Tween-20, 4 IA DKK1-coated beads (1.9×10⁶ beads) and 4 μl Cy5™detection antibody is prepared. A sample of 45 μl of this solution isdispensed per well into a 384-well FMAT black/clear bottom plate(Applied Biosystems). Fab-containing BEL extract (5 μl) is added to eachwell. The FMAT plates are incubated at room temperature overnight. Thenext morning the plates are analyzed in the 8200 Cellular DetectionSystem (Applied Biosystems).

Positive clones are obtained, and the heavy and light chain sequences ofclones yielding positive, specific signals in FMAT are analyzed. It isobserved that, 57 unique (non-redundant) anti-DKK1 clones are identifiedthat showed sufficient strong binding to human DKK1. These clones areexpressed, purified and tested for affinity and in functional assays.

Determination of Nanomolar Affinities Using Surface Plasmon Resonance

Using these clones, kinetic SPR analysis is performed on a CM5 chip(Biacore, Sweden) which had been coated with a density of ˜400 RU ofeither recombinant human DKK1, mouse DKK1 (R&D system), or cynomolgusDKK1 in 10 mM Na-acetate pH 4.5 using standard EDC-NHS amine couplingchemistry. A comparable amount of human serum albumin (HSA) isimmobilized on the reference flow cell. PBS (136 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1.76 mM KH2PO4 pH 7.4) is used as the running buffer. TheFab preparations are applied in concentration series of 16-500 nM at aflow rate of 20 μl/min. Association phase is set to 60 s anddissociation phase to 120 s. A summary of the affinities in nM to eachof human, mouse, and cynomolgus DKK1 determined by that method are shownin Table 1 herein.

TABLE 1 Affinities of selected Fabs to each of human, mouse, andcynomolgus KD [nM] KD [nM] KD [nM] human mouse cyno Antibody DKK1 DKK1*DKK1* MOR04470 3.2 ± 2.0 3.6 1.7 MOR04516 2.6 ± 0.7 2.4 1.9 MOR04454 3.2± 0.4 6 2.7 MOR04456 7.9 ± 0.9 11.6 8.1 MOR04461 7.6 ± 3.3 12.8 7.3MOR04455 1.6 ± 0.3 n.d. 1.5 *single measurement n.d.: not determined

Example 3 Identification of Anti-Human DKK1 Fab Candidates Inhibitingthe Wnt Antagonistic Activity of DKK1

The resulting 57 different DKK1-specific antibodies selected from theHuCAL GOLD® library are used to obtain purified antibody, which is thentested for potency to inhibit the Wnt antagonistic activity of humanDKK1. Of these, 17 antibody candidates are functionally active, as shownin FIGS. 2 and 4.

The functional activity of each of the HuCAL® Fabs is checked using aluciferase reporter gene assay. Twelve TCF/Lef binding sites are clonedupstream of the luciferase reporter gene rendering the luciferase geneTCF/Lef-responsive. The canonical Wnt proteins lead to a stabilizationof beta-catenin, thereby activating transcription of TCF/Lef andproducing the luciferase protein. Addition of DKK1 protein blocks Wntactivity and therefore also the transcription of the luciferase gene. Inconsequence, the luciferase levels produced by the respective cells areexpected to correlate with the potency of the selected Fabs to blockDKK1 action.

Stable TCF/Lef-Responsive Reporter Cell Line HEK293T/17-12xSTF

Bioassays are performed using the stable human embryonic kidney cellreporter cell line HEK293T/17-12xSTF. The cells are cultivated in DMEMhigh glucose medium (Invitrogen), containing 10% FCS (PAN orBioWhittaker) and 1 μg/ml puromycin (BD Biosciences), until 90%confluency is reached. The cells are then trypsinized, counted, anddiluted in culture medium without puromycin to a concentration of 4×10⁵cells per ml. Subsequently, the cells are seeded into a white,flat-bottom 96-well plate (Corning; 100 μl cell suspension per well) andincubated at 37° C. and 5% CO₂ over night. On the next day, the assaymedium is prepared: 500 ng/ml DKK1-APP is added to Wnt3a ConditionedMedium (CM). The anti-DKK1 HuCAL® Fabs (final concentration 20 μg/ml)and the goat anti-human DKK1 antibody (R&D Systems) used as a positivecontrol (final concentration 1.5 μg/ml) are diluted in CM.

A volume of 60 μl medium is removed from each well of the assay platewithout disturbing the adhering cells, and substituted by 60 μl of thetest antibody or control, diluted in CM. The cells are incubated foranother 24 h and 100 μl Bright-Glo luciferase reagent is added to eachwell. After 5 min incubation time, the luminescence is read in aluminometer (GenioPro, Tecan). The results obtained with 20 of the 57Fabs are shown in FIG. 2 herein. The extent of luciferase expressed is ameasure of the extent of antibody present.

Example 4 Quantitative Analysis of Binding Affinities: Determination ofAnti-Human DKK1 Fab Candidates that Inhibit the Wnt-AntagonisticActivity of DKK1 Affinity Determination

In order to further characterize the anti-DKK1 antibodies, the affinityto human, cynomolgus, and mouse DKK1 is determined. The recombinant DKK1protein is immobilized on a CM5 Biacore chip and the Fabs are applied inthe mobile phase in different concentrations. For a reliabledetermination of monovalent affinities only such Fab batches are usedfor Biacore measurements which showed ≧90% monomeric fraction in a sizeexclusion chromatography.

The summarized affinity data on human, mouse, and cynomolgus DKK1 isshown in Table 2. All 17 tested Fabs are found to have affinity to humanDKK1 below 100 nM. Further, nine of the clones produced antibodies withaffinities less than 10 nM. In all tested cases, the affinities forcynomolgus and mouse DKK1 are almost identical to those for human DKK1.

TABLE 2 Affinity data of selected Fabs on human, mouse, and cynomolgusKD [nM] KD [nM] KD [nM] cyno Antibody human DKK1 mouse DKK1* DKK1*MOR04480 1.0 ± 0.0 2 n.d. MOR04455 1.6 ± 0.3 n.d. 1.5 MOR04516 2.6 ± 0.72.4 1.9 MOR04470 3.2 ± 2.0 3.6 1.7 MOR04454 3.2 ± 0.4 6 2.7 MOR04483 5.5± 0.7 n.d. n.d. MOR04466 6.5 ± 2.1 n.d. n.d. MOR04461 7.6 ± 3.3 12.8 7.3MOR04456 7.9 ± 0.9 11.6 8.1 MOR04462 16.5 ± 2.1  n.d. n.d. MOR04447 22*n.d. n.d. MOR04469  36 ± 0.0 n.d. n.d. MOR04482  36 ± 5.7 n.d. n.d.MOR04468 41.5 ± 21.9 n.d. n.d. MOR04476 44* n.d. n.d. MOR04481 65* n.d.n.d. MOR04503 93* n.d. n.d. *single measurement n.d.: not determined

EC₅₀ Determination

The data showing the effective concentration for 50% inhibition for theclones of antibodies having the greatest affinity for DKK1 is shown inTable 3 herein. The data show that effective concentrations EC₅₀ rangefrom 39-95 nM, with a median value between 58 and 83 nM.

TABLE 3 Effective concentration for 50% inhibition of selected FabsLuciferase reporter assay; Antibody EC₅₀ [nM] MOR04470 58 MOR04516 42MOR04454 83 MOR04456 95 MOR04461 57 MOR04455 39

Example 5 Affinity Maturation of Selected Anti-DKK1 Fabs by ParallelExchange of LCDR3 and HCDR2 Cassettes

For optimizing the affinities of the antibodies described herein forDKK1 for a pool of parental Fab fragments, the LCDR3, framework 4 andthe constant region of the light chains (405 bp) of each parental Fab isremoved using BpiI and SphI, and is replaced by a repertoire ofdiversified LCDR3s together with framework 4 and the constant domain. Asample of 0.5 μg of the binder pool vector is ligated with a 3-foldmolar excess of the insert fragment carrying the diversified LCDR3s.

In a similar approach, the HCDR2 is diversified using the XhoI andBssHII sites, and the connecting framework regions are kept constant. Inorder to increase the cloning efficiency, the parental HCDR2 is replacedby a 590 bp stiffer sequence prior to the insertion of the diversifiedHCDR2 cassette.

Ligation mixtures of 11 different libraries are electroporated into 4 mlE. coli TOP10 F′ cells (Invitrogen, Carlsbad, Calif., USA), yieldingfrom 2×10⁷ to 2×10⁸ independent colonies. Amplification of the librariesis performed as previously described (Rauchenberger et al., 2003 J BiolChem. 278(40):38194-38205). For quality control, several clones perlibrary are randomly picked and sequenced (SequiServe, Vaterstetten,Germany) using primers CFR84 (VL) and OCAL_Seq_Hp (VH).

Selection of Candidates for Affinity Maturation

Six selected maturation candidates (“parental Fabs”) are selected byhaving been characterized as having the following properties: affinitiesto human DKK1 less than 10 nM, with significant cross-reactivity tocynomolgus and mouse DKK1, EC₅₀ less than 100 nM, and good to moderateFab expression levels in E. coli and lack of aggregation after Fabpurification.

During the course of the affinity measurements, it became evident thatMOR04480 is highly unstable at high dilutions. For this reason, MOR04480is omitted from the list of maturation candidates albeit having thehighest affinity (1 nM) and the best EC₅₀ (7 nM) of all tested Fabs.MOR04483 had a high affinity of 5.5 nM to human DKK1 but is shown to becrossreactive to mouse DKK1, and MOR04453 contained a high proportion ofFab aggregates after purification. Therefore, these two antibodies arealso excluded from the maturation.

After careful evaluation of all available data, six maturationcandidates (MOR04454, MOR04455, MOR04456, MOR04461, MOR04470, andMOR04516) are selected. The properties of these candidates are listed inTable 4 herein.

TABLE 4 Properties of selected Fabs KD [nM] KD [nM] KD [nM] Cross- Fabhuman mouse cyno EC50 reactivity expression Size exclusion Antibody DKK1DKK1* DKK1* [nM] mouse [mg/1] chromatography MOR04470 3.2 ± 2.0 3.6 1.758 ++ 32.8 # MOR04516 2.6 ± 0.7 2.4 1.9 42 ++ 1.5 # MOR04454 3.2 ± 0.4 62.7 83 ++ 17.8 # MOR04456 7.9 ± 0.9 11.6 8.1 95 ++ 10.7 # MOR04461 7.6 ±3.3 12.8 7.3 57 ++ 12 # MOR04455 1.6 ± 0.3 n.d. 1.5 39 ++ 9 # *singlemeasurement n.d.: not determined # monomeric portion > 90%

Generation of Selected Fab Libraries for Affinity Maturation

In order to obtain clones having increased affinity and inhibitoryactivity of the anti-DKK1 antibodies, the selected Fab clones MOR04454,MOR04455, MOR04456, MOR04461, MOR04470, and MOR4516 shown in theprevious example are subjected to further rounds of diversification andselection, a process known as affinity maturation.

For this purpose, CDR regions are diversified using corresponding LCDR3and HCDR2 maturation cassettes pre-built by trinucleotide mutagenesis(Virnekäs et al., 1994 Nucleic Acids Res. 22:5600-5607; Nagy et al.,2002 Nature Medicine 8:801-807). Table 5 herein shows the LCDR3sequences for the parental clones MOR04454, MOR04455, MOR04456, MOR061,MOR04470 and MOR4516.

TABLE 5 LCDR3 sequences for selected Fabs LCDR3 Antibody VL SequenceSEQ ID NO: MOR04454 K1 LQYYGMPP 21 MOR04455 K1 QQYDSIPM 22 MOR04456 K3QQYGDEPI 23 MOR04470 L2 QSYASGNTKV 25 MOR04461 L2 STWDMTVDF 24 MOR04516L1 ASFDMGSPNV 26

Table 6 herein shows the HCDR2 sequences for the parental clonesMOR04454, MOR04455, MOR04456, MOR061, MOR04470 and MOR4516.

TABLE 6 HCDR2 sequences for selected Fabs Antibody VH HCDR2 SequenceSEQ ID NO: MOR04454 H3 DGSHMDKPPGYVFAF 2 MOR04455 H3 HYMDH 3 MOR04456 H3TIYMDY 4 MOR04461 H3 MGIDLDY 5 MOR04470 H3 HGIDFDH 6 MOR04516 H5GIPFRMRGFDY 7

Fab fragments from expression vector pMORPH®X9_Fab_FH are subcloned intothe phagemid vector pMORPH®25 (see U.S. Pat. No. 6,753,136). This vectorprovides the phage protein pIII fused N-terminally to a cysteine residueas well as a C-terminal cysteine to the Fd antibody chain and thusallows disulfide-linked display of the respective Fab fragments on thephage surface. Two different strategies are applied in parallel tooptimize both the affinity and the efficacy of the parental Fabs.

Five phage antibody Fab libraries are generated in which the LCDR3 offive of the six parental clones is replaced by a repertoire ofindividual light chain CDR3 sequences. The LCDR3 maturation of MOR04454is not performed, as this clone has an additional BpiI restriction sitein one of the CDR regions and the BpiI restriction enzyme is used forthe library cloning procedure.)

In parallel, the HCDR2 region of each parental clone is replaced by arepertoire of individual heavy chain CDR2 sequences. Each parental Fabis excised and replaced for a 590 bp stuffer. This DNA stufferfacilitates the separation of single digested from double digestedvector bands and reduces the background of the high-affinity parentalFabs during the maturation pannings. In a subsequent step, the stifferis excised from the Fab-encoding plasmids of each parental clone andreplaced for the highly diversified HCDR2 maturation cassette.

Large affinity maturation libraries of more than 2×10⁷ members aregenerated by standard cloning procedures, and the diversified clones aretransformed into electro-competent E. coli TOP10F′ cells (Invitrogen).Fab-presenting phages are prepared as described in Example 1 above.

Four maturation pools are built in order to facilitate the subsequentselection process: pool 1a consisted of the MOR04470, and MOR04516 LCDR3libraries; pool 1b consisted of the MOR04470, and MOR04516 HCDR2libraries; pool 2a consisted of the MOR04454, MOR04455, MOR04456, andMOR04461 LCDR3 libraries; and pool 2b consisted of the MOR04454,MOR04455, MOR04456, and MOR04461 HCDR2 libraries.

For each pool the panning is performed in solution using decreasingamounts of His-Strep-tagged DKK1 and phage-antigen capturing byStrep-Tactin beads. In parallel, each pool is applied in pannings usingdecreasing amounts of biotinylated DKK1, which is captured ontoNeutravidin-coated plates. In order to increase the panning stringencyand to select for improved off rates, competition with purified parentalFabs as well as unlabeled antigen is performed during prolongedincubation periods.

Immediately after the pannings the enriched phagemid pools are subclonedinto the pMORPH®X9_FH expression vector. About 2300 single clones arepicked, and the Fabs are induced with IPTG.

Maturation Panning Strategies

Panning procedures using the four antibody pools are performed withHis-Strep-tagged DKK1 and with biotinylated His-Strep-tagged DKK1 insolution for two or three rounds, respectively. For each of the panningstrategies, competition with the purified parental Fab proteins or withunlabeled APP-tagged DKK1, as well as low antigen concentrations andextensive washing, are used to increase stringency.

The solution panning on unlabeled His-Strep-tagged DKK1 is performedover two selection rounds mainly according to the standard protocoldescribed in Example 1. Exceptions to these procedures are theapplication of reduced amounts of antigen (decreasing from 5 nM down to1 nM), the high stringency of the washing procedure either withcompetitor or without, and prolonged incubation periods ofantibody-phages together with the antigen.

For the first selection round using biotinylated DKK1, the wells of aNeutravidin plate are washed two times with 300 μl PBS. The wells areblocked with 2×ChemiBLOCKER (Chemicon, Temecula, Calif.) diluted 1:1 inPBS (Blocking Buffer). Prior to the selections, the HuCAL GOLD® phagesare also blocked with one volume Blocking Buffer containing 0.1%Tween-20 for 30 min at room temperature. The blocked phage preparationsare transferred in 100 μl aliquots to the wells of a Neutravidin-coatedplate for 30 min at room temperature. This pre-adsorption step isrepeated once. Blocked and pre-cleared phage preparations are incubatedwith 5 nM biotinylated DKK1 for 2 h at 22° C. on a rotating wheel.Parental Fab, APP-DKK1 or no competitor is added and the samples areincubated overnight at 4° C. on a rotating wheel.

Antigen-phage complexes are captured in the wells of a Neutravidin platefor 20 min at room temperature. After extensive washing steps, boundphage particles are eluted by addition of 200 μl of 20 mM DTT in 10 mMTris pH 8.0 per well for 10 min at room temperature. The eluate isremoved and added to 14 ml E. coli TG1 cells grown to an OD_(600nm) of0.6-0.8. The wells are rinsed once with 200 μl PBS and this solution isalso added to the E. coli TG1 cells. Phage infection of E. coli isallowed for 45 min at 37° C. without shaking. After centrifugation for10 min at 5000 rpm, the bacterial pellets are each resuspended in 500 μl2×YT medium, plated onto 2×YT-CG agar plates and incubated overnight at30° C. The colonies are harvested by scraping from the surface of theplates and the phage particles are rescued and amplified as describedabove.

The second and third round of the selection are performed as describedabove for the first round of selection, excepted that washing conditionsare more stringent and antigen concentrations are 1 and 0.1 nM,respectively.

Electrochemiluminescence (BioVeris)-Based Binding Analysis of DKK1Binding Fabs

For the detection of affinity-improved, DKK1-specific antibody fragmentsin E. coli lysates (BEL extracts), a BioVeris M-384 SERIES® Workstation(BioVeris Europe, Witney, Oxfordshire, UK), is used. The assay iscarried out in 96-well polypropylene microtiter plates and PBSsupplemented with 0.5% BSA and 0.02% Tween-20 as the assay buffer.Biotinylated human DKK1 is immobilized on M-280 Streptavidinparamagnetic beads (Dynal) according to the instructions of thesupplier. A 1:25 dilution of the bead stock solution is added per well.Samples of 100 μl diluted BEL extract and beads are incubated overnightat room temperature on a shaker. For detection, anti-human (Fab)′2(Dianova) labelled with BV-tag™ according to instructions of thesupplier (BioVeris Europe, Witney, Oxfordshire, UK) is used.

A set of about 2300 randomly picked clones are analyzed by the methoddescribed above. A subset of 160 clones giving the highest values ischosen for further analysis in solution equilibrium titration.

Determination of Picomolar Affinities Using Solution EquilibriumTitration (SET)

For K_(D) determination, monomer fractions (at least 90% monomercontent, analyzed by analytical SEC; Superdex75, Amersham Pharmacia) ofFab are used. Electrochemiluminescence (ECL) based affinitydetermination in solution and data evaluation are basically performed asdescribed by Haenel et al., 2005. A constant amount of Fab isequilibrated with different concentrations (serial 3^(n) dilutions) ofhuman DKK1 (4 nM starting concentration) in solution. Biotinylated humanDKK1 coupled to paramagnetic beads (M-280 Streptavidin, Dynal), andBV-tag™ (BioVeris Europe, Witney, Oxfordshire, UK) labelled anti-human(Fab)′₂ (Dianova) is added and the mixture incubated for 30 min.Subsequently, the concentration of unbound Fab is quantified by ECLdetection using the M-SERIES® 384 analyzer (BioVeris Europe).

For this purpose, 160 single clones are selected and purified by Ni-NTAAgarose in the μg scale. Preliminary affinities are determined by4-point solution equilibrium titration (SET) in BioVeris. From thesedata, 20 clones showing affinities are selected. These Fabs are purifiedin the mg scale. MOR04950 is excluded from affinity determination andfurther evaluation due to partial aggregation of the Fab which isdetected in size exclusion chromatography. Final affinities aredetermined from two independent batches of each Fab clone using an8-point SET measurement and human, mouse, and cynomolgus DKK1.

Affinity determination to mouse and cynomolgus DKK1 is done essentiallyas described above using mouse DKK1 (R&D Systems) and cynomolgus DKK1 asanalyte in solution instead of human DKK1. For detection of free Fab,biotinylated human DKK1 coupled to paramagnetic beads is used.Affinities are calculated according to Haenel et al., 2005 Anal Biochem339.1:182-184.

Using the assay conditions described above, the affinities for theaffinity-optimized anti-DKK1 Fabs are determined in solution. Affinitiesare determined for MOR04910 and MOR04946, with K_(D)s below 30 pM tohuman DKK1 and between 36 and 42 pM to mouse and cynomolgus DKK1.Further seven antibodies showed affinities below 100 pM to all threeantigens. Clone MOR04950 did not show binding to the biotinylated DKK1.The affinities are summarized in Table 7 herein.

TABLE 7 Affinities of Fabs Affinity [pM]: solution equilibriumtitration* Antibody Human DKK1 Cyno DKK1 Mouse DKK1 MOR04913 38 ± 8  51± 10  44 ± 48 MOR04946 25 ± 9 36 ± 9  41 ± 15 MOR04907 114 ± 7  170 ± 35167 ± 68 MOR04945  69 ± 18  80 ± 31  74 ± 21 MOR04914 94 ± 5 140 ± 3  200 ± 261 MOR04920  53 ± 37  50 ± 20  40 ± 30 MOR04954 110 ± 54 112 ±25 36 ± 4 MOR04952 100 ± 12  58 ± 17  71 ± 34 MOR04948 57 ± 8  67 ± 17 65 ± 25 MOR04910 24 ± 3  37 ± 11  42 ± 38 MOR04921 high batch-to batchvariation MOR04947  32 ± 19  58 ± 35  30 ± 17 MOR04951  136 ± 120 62 ± 2132 ± 90 MOR04918 110-690 236 ± 23 550-3144 MOR04919  64 # 132 # 111 #MOR04949  30 ± 23  55 ± 35  65 ± 36 MOR04922  90 #  70 #  50 # MOR04911140 # 190 # 650 # MOR04950 no binding *at least two independentmeasurements performed from two different Fab baches # singlemeasurement only

Example 6 Characterization of Affinity-Optimized Anti-Human DKK1 FabsEnzyme Linked Immuno Sorbent Assay (ELISA) Techniques

Binding specificity of the matured Fabs in the presence of 50% humanserum (HS) is determined. Serial dilutions of human recombinant,biotinylated DKK1 in TBS are coated onto Neutravidin microtiter platesfor 2 h at room temperature, from 8 ng DKK1 per well to a concentrationof 125 ng DKK1 per well. After coating of the antigen, wells are blockedwith TBS/0.05% Tween (TBS-T) supplemented with 1% BSA for 1 h at roomtemperature. Purified Fabs described above are diluted either in TBS/4%BSA or TBS/50% HS at a final concentration of 1 μg/ml, added to thecoated and blocked wells and the plates are incubated for 1 h at roomtemperature. For detection, an anti-FLAG alkaline phosphatase(AP)-conjugated antibody (1:5000 dilution in TBST) and the fluorogenicsubstrate AttoPhos (Roche) are used. After each incubation, the wells ofthe microtiter plates are washed with TBST five times, except after thefinal incubation step with the labeled secondary antibody when wells arewashed three times.

The fluorescence is measured in a TECAN Spectrafluor plate reader.Exemplary binding curves are shown in FIG. 3 herein. Table 8 summarizesthe binding activity of the optimized anti-DKK1 Fabs in presence of 50%human serum compared to binding activity in 4% BSA, which ranges from83% to 100%. The median value is found to be 93%, thus the anti-DKK1Fabs are found to fully bind to target in the presence of human serum.

TABLE 8 Binding activity of Fabs Binding activity w/50% Antibody Humanserum vs 4% BSA (%) MOR04945 89 ± 6 MOR04910 84 ± 4 MOR04946 93 ± 2MOR04913 89 ± 9 MOR04920 90 ± 7 MOR04948 100 ± 15 MOR04952 100 ± 5 MOR04921 93* MOR04914 83* MOR04907 >83*   MOR04954 >83*   MOR04937 95**single measurement

Luciferase Reporter Cell Assay in Presence of Human Serum Using the U2OSCell Line

For a further determination of binding specificity of the optimizedanti-DKK1 Fabs, the luciferase reporter cell assay is repeated inpresence of 15% human serum using the osteosarcoma cell line U2OS. TheU2OS cells (ATCC No. HTB-96) are grown according to the provider'sprotocol (ATCC, Manassas, Va., USA). The cells are trypsinized, counted,and diluted in culture medium (McCoy's 5a/10% FCS) to a concentration of2×10⁵ cells/ml. For each 2×10⁴ cells, a solution is prepared that is amixture of 0.075 μg pTA-LUC-12xSuperTopFlash and 0.004 μg phRL-SV40.These are mixed in a final volume of 9.8 μl OPTI-MEM. Then 0.2 μl FuGENE6 Transfection Reagent (Roche, Mannheim, Germany) is added. Thistransfection mix is briefly incubated and then mixed with the previouslyprepared cells. Subsequently, the cells are seeded in 100 μl per well ofa white flat-bottomed 96-well cell culture dish and incubated at 37° C.and 5% CO₂ over night. The next day, 75 μl medium are removed from eachwell of the assay plate and substituted by 10 μl of HuCAL® Fabantibodies dilutions from (10 to 0.01 μg/ml diluted in serum-freeculture medium), 15 μl of either 70% FCS or Human Serum, and 50 μl ofthe Wnt3a Conditioned Medium, containing 600 ng/ml DKK1-APP is added toeach well.

For a negative control, serum-free medium is added instead of antibodydilutions. In order to obtain a maximum luciferase signal, controlscontaining 10 μl serum-free medium instead of antibody dilutions and 50μl Wnt3a CM without DKK1-APP are added. After 24 h incubation at 37° C.,5% CO₂, the luminescence is measured with the Dual-Glo Luciferase AssaySystem (Promega, Madison, Wis., USA) according to the manufacturer'sinstructions.

Table 9 shows the inhibitory activity of the optimized anti-DKK1 Fabs inpresence of 15% human serum (compared to the inhibitory activity in 15%FCS). The data shows that the activity obtained in the presence of serumranged from 26% to 90%, with a median value of 70-74%. These data showthat clones of the anti-DKK1 Fabs are obtained that function in thepresence of human serum.

TABLE 9 Inhibitory activity of Fabs Luciferase reporter assay w/oKremen; Activity in 15% Antibody Human serum (%) MOR04945 74% MOR0491090% MOR04946 60% MOR04913 88% MOR04920 75% MOR04948 70% MOR04952 60%MOR04921 26% MOR04914 80% MOR04907 54% MOR04954 55% MOR04937 84%

EC₅₀ Determination of Affinity-Optimized Anti-DKK1 Fabs by LuciferaseReporter Cell Assay

The test of the affinity-improved Fabs in the standard Wnt3a-dependentTCF/LEF luc reporter assay used 10 nM DKK1 in order to obtain inhibitionof the luciferase expression. It is seen that EC₅₀ values could not begenerated by this method as the sensitivity of the assay is too low.This is indicated by very steep inhibition curves and similar EC₅₀values for all Fabs tested as seen in FIG. 4 herein.

An improved version of the TCF/LEF luc reporter assay is developed. DKK1binds to the Kremen-1 and -2 transmembrane proteins and this interactionleads to a strong synergistic inhibition of Wnt signaling (Mao et al.2002 Nature: 417: 664-67). Therefore, Kremen cDNA is co-transfected withthe TCF/LEF luc reporter assay. The resulting Wnt3a-dependent reporterassay showed highly improved sensitivity to DKK1, mediated byco-expression of the Kremen co-receptor protein. In this assay, 0.33 nMDKK1 is sufficient to induce full inhibition of Wnt signaling. The Fabtitrations (at ten concentrations) are repeated using 0.33 nM DKK1, andyielded sigmoid inhibition curves (FIG. 5 herein) from which EC₅₀ valuescould be calculated.

The affinity-optimized anti-DKK1 Fabs are thereby analyzed with respectto EC₅₀ as described above. As shown in Table 10 herein, the EC₅₀ valuesobtained by this method ranged from 0.2 nM to 5.6 nM.

TABLE 10 EC₅₀ of Fabs TCF/LEF luc assay w/Kremen: EC50 Antibody [nM]MOR04913 0.5 MOR04946 0.2 MOR04907 0.9 MOR04945 0.5 MOR04914 1.1MOR04920 1.8 MOR04954 1.3 MOR04952 0.6 MOR04948 0.5 MOR04910 0.3MOR04921 0.7 MOR04947 1.7 MOR04951 2.2 MOR04919 2.7 MOR04949 5.3MOR04922 2 MOR04911 5.6

Sequence Analysis of the Affinity-Optimized Fabs

The nucleotide sequences of the heavy and V_(L) regions (V_(H) andV_(L)) of all twenty Fabs are determined Amino acid sequences of thecomplementarity determining regions (CDRs) are listed in Table 11A andTable 11B herein

TABLE 11A Amino acid sequences of Heavy Chain CDR's SEQ ID SEQ ID SEQ IDNo. No. No. HCDR HCDR HCDR Antibody VH HCDR1 1 HCDR2 2 HCDR3 3 P MOR0445VH GFTFSSYGMS  3 WVSGISGSGSYTYYADSVKG  3 HYMDH  3 5 3 1 MOR0491 VHGFTFSSYGMS 49 WVSGISERGVYIFYADSVKG 57 HYMDH 65 8 3 P MOR0445 VHGFTFNNYGMT  4 WVSGISGSGSYTYYADSVKG  4 TIYMDY  4 6 3 2 MOR0490 VHGFTFNNYGMT  8 WVSGISGSGSYTYYADSVKG  8 TIYMDY  8 7 3 3 MOR0494 VHGFTFNNYGMT 10 WVSGISGSGSYTYYADSVKG 10 TIYMDY 10 6 3 4 MOR0494 VHGFTFNNYGMT 50 WVSGISGSGSYTYYADSVKG 58 TIYMDY 66 9 3 5 MOR0491 VHGFTFNNYGMT  9 WVSGISGSGSYTYYADSVKG  9 TIYMDY  9 3 3 P MOR0446 VHGFTFSSYWM  5 WVSGISYSGSNTHYADSVKG  5 MGIDLDY  5 1 3 S 6 MOR0491 VHGFTFSSYWM 51 WVSDIEHKRRAGGATSYAASVK 59 MGIDLDY 67 1 3 S G 7 MOR0492 VHGFTFSSYWM 52 WVSMIEHKTRGGTTDYAAPVKG 60 MGIDLDY 68 2 3 S 8 MOR0491 VHGFTFSSYWM 11 WVSGISYSGSNTHYADSVKG 11 MGIDLDY 11 0 3 S 9 MOR0494 VHGFTFSSYWM 13 WVSGISYSGSNTHYADSVKG 13 MGIDLDY 13 8 3 S 1 MOR0491 VHGFTFSSYWM 53 WVSGISYSGSNTHYADSVKG 61 MGIDLDY 69 0 9 3 S 1 MOR0492 VHGFTFSSYWM 12 WVSGISYSGSNTHYADSVKG 12 MGIDLDY 12 1 1 3 S P MOR0447 VHGFTFSSYWM  6 WVSVISSDSSSTYYADSVKG  6 HGIDFDH  6 0 3 S 1 MOR0491 VHGFTFSSYWM 14 WVSVISSDSSSTYYADSVKG 14 HGIDFDH 14 2 4 3 S 1 MOR0494 VHGFTFSSYWM 16 WVSVISSDSSSTYYADSVKG 16 HGIDFDH 16 3 5 3 S 1 MOR0495 VHGFTFSSYWM 54 WVSVISSDSSSTYYADSVKG 62 HGIDFDH 70 4 1 3 S 1 MOR0495 VHGFTFSSYWM 55 WVSVISSDSSSTYYADSVKG 63 HGIDFDH 71 5 2 3 S 1 MOR0495 VHGFTFSSYWM 56 WVSVIEHKSFGSATFYAASVKG 64 HGIDFDH 72 6 0 3 S 1 MOR0495 VHGFTFSSYWM 18 WVSVIEHKDKGGTTYYAASVKG 18 HGIDFDH 18 7 4 3 S 1 MOR0492 VHGFTFSSYWM 15 WVSSIEHKDAGYTTWYAAGVKG 15 HGIDFDH 15 8 0 3 S P MOR0451 VHGYSFTNYYIG  7 WMGIIYPTDSYTNYSPSFQG  7 GIPFRMRGFD  7 6 5 Y 1 MOR0494 VHGYSFTNYYIG 19 WMGIIYPGTSYTIYSPSFGQ 19 GIPFRMRGFD 19 9 7 5 Y

TABLE 11B Amino acid sequences of Light Chain CDR's MOR SEQ SEQ SEQAntibody ID ID ID No. V_(L) HCDR1 No.   HCDR2 No. hCDR3 No. Comment  P04455 K1 RASQDISNYLH  22 LLIYGASNLQS  22 QQYDSIPM  22  1 04918 K1RASQDISNYLH  73 LLIYGASNLQS  81 QQYDSIPM  89  P 04456 K3 RASQNLFSPYLA 23 LLIYGASNRAT  23 QQYGDEPI  23  2 04907 K3 RASQNLFSPYLA  27LLIYGASNRAT  27 QQYLSLPT  27  3 04946 K3 RASQNLFSPYLA  29 LLIYGASNRAT 29 QQYLTLPL  29  4 04949 K3 RASQNLFSPYLA  74 LLIYGASNRAT  82 QQYLFPL 90  5 04913 K3 RASQNLFSPYLA  28 LLIYGASNRAT  28 QQYMTLPL  28FW4 mutation  P 04461 L2 TGTSSDVGGFNYVS  24 LMIHDGSNRPS  24 STWDMTVDF 24  6 04911 L2 TGTSSDVGGFNYVS  75 LMIHDGSNRPS  83 STWDMTVDF  91  704922 L2 TGTSSDVGGFNYVS  76 LMIHDGSNRPS  84 STWDMTVDF  92  8 04910 L2TGTSSDVGGFNYVS  30 LMIHDGSNRPS  30 QSWDVSPITA  30  9 04948 L2TGTSSDVGGFNYVS  32 LMIHDGSNRPS  32 QTWDSLSFF  32 10 04919 L2TGTSSDVGGFNYVS  77 LMIHDGSNRPS  85 QSWGVGPGGF  93 11 04921 L2TGTSSDVGGFNYVS  31 LMIHDGSNRPS  31 QTWATSPLSS  31  P 04470 L2TGTSSDLGGYNYVS  25 LMIYDVNNRPS  25 QSYASGNTKV  25 12 04914 L2TGTSSDLGGYNYVS  33 LMIYDVNNRPS  33 QSYTYTPISP  33 13 04945 L2TGTSSDLGGYNYVS  35 LMIYDVNNRPS  35 QTYDQIKLSA  35 14 04951 L2TGTSSDLGGYNYVS  78 LMIYDVNNRPS  86 QSYDPFLDVV  94 15 04952 L2TGTSSDLGGYNYVS  79 LMIYDVNNRPS  87 QSYDSPTDSV  95 16 04950 L2TGTSSDLGGYNYVS  80 LMIYDVNNRPS  88 QSYASGNTKV  96 17 04954 L2TGTSSDLGGYNYVS  37 LMIYDVNNRPS  37 QSYASGNTKV  37 18 04920 L2TGTSSDLGGYNYVS  34 LMIYDVNNRPS  34 QSYASGNTKV  34 HCDR2 point mutation P 04516 L1 SGSSSNIGSSFVN  26 LLIGNNSNRPS  26 ASFDMGSPNV  26Potential N- glycosyl sites 19 04947 L1 SGSSSNIGSSFVN  38 LLIGNNSNRPS 38 ASFDMGSPNV  38 Potential N- glycosyl site consensus1 RASQxxxxxYx 131LLIYGASNxxx 132 QQYxxxPx 133 consensus2 TGTSSDVGGFNYVS 134 LMIxDxxNRPS135 xxWDxxxxx 136

The sequence analysis showed that five of the six parental (P) Fabsyielded affinity-improved successors. MOR04461 and MOR04470 could beoptimized in HCDR2 as well as in LCDR3. No optimized successors ofMOR04454 are obtained. In addition, high homology appears betweendisparate parent antibodies, as shown in consensus1 and consensus2sequences for the various CDRs in Table 11B Similar consensus sequencesmay be provided for parent sequences shown in Table 10A using methodswell known to one skilled in the art.

In addition, it is determined that MOR04920 has a mutation in the HCDR2region (a Ser residue to a Gly at pos. 73 according to the numberingscheme published by Honegger and Pluckthun, 2001 J Mol Biol309.3:657-670 thus deviating from the HuCAL® design.

MOR04913 is shown to have a point mutation in framework 4 of the kappalight chain (Lys to Asn exchange at position 148). As this position isnot expected to have an effect on the binding properties of the antibodythe mutation is reverted back to the germline/HuCAL® composition duringIgG conversion, yielding antibody MOR05145.

MOR04947 has a potential glycosylation site in LCDR2. This site is notremoved as MOR04947 is selected only as one of the back-up candidates.

Example 7 Production of HuCAL Immunoglobulins

Conversion into the IgG Format

In order to express full length immunoglobulin (Ig), variable domainfragments of heavy (V_(H)) and light chains (V_(L)) are subcloned fromthe pMORPH®X9_FH Fab expression vectors either into the pMORPH®_h_Ig orthe pMORPH®2_h_Ig vector series for human IgG1 and human IgG4.Alternative vectors may be used for human IgG2. Restriction enzymesEcoRI, MfeI, and BlpI are used for subcloning of the V_(H) domainfragment into pMORPH®_h_IgG1 and pMORPH®_h_IgG4. Restriction enzymesMfeI and BlpI are used for subcloning of the V_(H) domain fragment intopMORPH®2_h_IgG1f and pMORPH®2_h_IgG4. Subcloning of the V_(L) domainfragment into pMORPH®_h_Igκ and pMORPH®2_h_Igκ is performed using theEcoRV and BsiWI sites, whereas subcloning into pMORPH®_h_Igλ andpMORPH®2_h_Igλ2 is done using EcoRV and HpaI.

Transient Expression and Purification of Human IgG

HEK293 cells are transfected with an equimolar amount of IgG heavy andlight chain expression vectors. On days 4 or 5 after transfection, thecell culture supernatant is harvested. After adjusting the pH of thesupernatant to 8.0 and sterile filtration, the solution is subjected tostandard protein A column chromatography (Poros 20A, PE Biosystems).

Conversion of Parental Fabs into the IgG Formats

In parallel to the start of the affinity maturation, MOR04454, MOR04456,and MOR04470 are cloned into the pMORPH®_h_IgG1 and pMORPH®_h_IgG4expression vectors. Alternative constructs may be used for creation ofIgG2 expression vectors. Small scale expression is performed bytransient transfection of HEK293 cells and the full lengthimmunoglobulins are purified from the cell culture supernatant.

The data show by size exclusion chromatography that the antibodies arein monomeric form. Testing in the Wnt3a-dependent reporter assay provedthat the proteins are functional.

Example 8 Amino Acid Sequences and Nucleotide Sequences of GenesOptimized for Expression

To increase mammalian expression, changes are introduced into the heavyand the light chains of Fabs herein for optimization of codon usage forexpression in a cell. It is known that several negatively cis-actingmotifs decrease expression in mammals. The optimization process hereinremoves negative cis-acting sites (such as splice sites or poly(A)signals) which negatively influence expression. The optimization processherein further enriches GC content, to prolong mRNA half-life.

Variable light and heavy chain regions are optimized using a clone of aFab, MOR04945 (full length light chain parental nucleotide sequence isSEQ ID NO: 98 and full length heavy chain parental nucleotide sequenceis SEQ ID NO: 102), isolated herein by selection with phage display.Then the nucleotide sequences encoding each of the entire light andheavy chains of this and other clones are each optimized using theseprocedures.

Optimization Process for V_(H) and V_(L) Chains of MOR04945

For optimizing the nucleotide sequence and amino acid sequence of eachof the V_(L) and V_(H) chains for expression in mammalian cells, thecodon usage is adapted to the codon bias of mammalian genes. Inaddition, regions of very high (>80%) or very low (<30%) GC content arereduced or eliminated where possible. Alternatively, optimization forexpression in bacteria, yeast or baculovirus would entail adapting codonusage biased for their respective genes.

During the optimization process for mammalian expression, the followingcis-acting sequence motifs are avoided: internal TATA-boxes, chi-sitesand ribosomal entry sites, AT-rich or GC-rich sequence stretches, RNAinstability motif (ARE) sequence elements, inhibitory RNA sequenceelements (INS), cAMP responsive (CRS) sequence elements, repeatsequences and RNA secondary structures, splice donor and acceptor sitesincluding cryptic sites, and branch points. Except as indicated,introduction of MluI and HindIII sites is avoided in the process ofoptimizing the nucleotide sequence of the V_(L) chain. Except asindicated, introduction of MlyI and BstEII sites is avoided in theprocess of optimizing the nucleotide sequence of the V_(H) chain.

Amino Acid Sequences of V_(H) and V_(L) Chains of MOR04945 Optimized forExpression

Codon usage is adapted to that of mammals to enable higher and morestable expression rates in a mammalian cell for the resulting optimizedamino acid sequences for the V_(H) and V_(L) chains of the cloneMOR04945 described above. See Example 5.

Table 12A below shows the sense and anti-sense (AS) nucleotide sequencesof the variable light chain (SEQ ID NO: 120) and the resulting variablelight chain amino acid (designated AA) sequence (SEQ ID NO: 118) asoptimized for expression.

TABLE 12A Nucleotide sense and antisense sequences,  and amino acid sequences chain of V_(H) optimized for expression

Table 12B below shows sense and anti-sense variable heavy chainnucleotide sequences (SEQ ID NO: 121) and the resulting variable heavychain amino acid (designated AA) sequence (SEQ ID NO: 119) as optimizedfor expression.

TABLE 12B Nucleotide sense and antisense sequences,  and amino acid sequences of V_(H) chain optimized for expression

Pre- and post-optimization charts may provide the percentages ofsequence codons for each of the parental sequences and optimized genesrespectively, and analyses the quality class of the respectingnucleotide sequences encoding the V_(H) and V_(L) chains. Quality valueas used herein means that the most frequent codon used for a given aminoacid in the desired expression system is set as 100, and the remainingcodons are scaled accordingly to frequency of usage. (Sharp, P.M., Li,W. H., Nucleic Acids Res. 15 (3), 1987).

Further, the codon adaptation index (CAI) is a number that describes howwell the codons of the nucleotide sequence match the codon usagepreference of the target organism. The maximum value of CAI is set to1.0, thus a CAI of >0.9 is considered as enabling high expression. TheCAI for the V_(L) chain prior to optimization is found to be 0.73, andafter optimization, the CAI is determined to be 0.95. Similarly, the CAIfor the V_(H) chain prior to optimization is found to be 0.74, and afteroptimization, is determined to be 0.98 in optimized constructs, the GCcontent in the V_(L) chain is increased from 51% for the parent sequenceof MOR04945 to 62% for the optimized sequence derived from MOR04945. Asshown in FIGS. 9A and 9B, the GC content in the V_(H) chain is increasedfrom 54% for the parent sequence of MOR04945 to 64% for the optimizedderivative of MOR04945.

Optimization for Expression of Full Length Light Chains and Heavy Chainsof MOR04910, MOR04945, MOR04946, and MOR05145

The optimization process is applied to each of the parent full lengthnucleotide sequences of the light chains of MOR04910 (SEQ ID NO: 97),MOR04945 (SEQ ID NO: 98), MOR04946 (SEQ ID NO: 99), and MOR05145 (SEQ IDNO: 100) and the parent full length nucleotide sequences of the heavychains of MOR04910 (SEQ ID NO: 101), MOR04945 (SEQ ID NO: 102), MOR04946(SEQ ID NO: 103), and MOR05145 (SEQ ID NO: 103).

The optimization process is used to construct each of the followinglight chain nucleotide sequences associated with the parent clonenumbers: for clone MOR04910 the optimized nucleotide sequence is SEQ IDNO: 104; for clone MOR04945 the optimized nucleotide sequence is SEQ IDNO: 105; for clone MOR04946 the optimized nucleotide sequence is SEQ IDNO: 106, and for clone MOR05145 the optimized nucleotide sequence is SEQID NO: 107. Further, the optimization process is used to construct eachof the following heavy chain nucleotide sequences associated with theparent clone numbers: for clone MOR04910 the optimized nucleotidesequence is SEQ ID NO: 108; for clone MOR04945 the optimized nucleotidesequence is SEQ ID NO: 109; for clone MOR04946 the optimized nucleotidesequence is SEQ ID NO: 110; and for clone MOR05145 the optimizednucleotide sequence is SEQ ID NO: 110.

The optimized light chain nucleotide sequences are associated with thefollowing optimized light chain amino acid sequences: for clone MOR04910the optimized amino acid sequence is SEQ ID NO: 111; for clone MOR04945the optimized amino acid sequence is SEQ ID NO: 112; for clone MOR04946the optimized amino acid sequence is SEQ ID NO: 113; and for cloneMOR05145 the optimized amino acid sequence is SEQ ID NO: 114. Theoptimized heavy chain nucleotide sequences are associated with thefollowing optimized heavy chain amino acid sequences: for clone MOR04910the optimized amino acid sequence is SEQ ID NO: 115; for clone MOR04945the optimized amino acid sequence is SEQ ID NO: 116; for clone MOR04946the optimized amino acid sequence is SEQ ID NO: 117; and for cloneMOR05145 the optimized amino acid sequence is SEQ ID NO: 117.

A listing of nucleotide and polypeptide sequences of contemplated fulllength light and heavy chain sequences are provided in Table 13A andTable 13B. Table 13A provides optimized nucleotide sequences and thepolypeptides encoded by them. These nucleotide sequences are optimizedto remove latent splice sites that are recognized in mammalianexpression systems. Table 13B provides the parental nucleotide sequencesfor the clones listed in Table 13A.

TABLE 13A Light Chain (LC) and Heavy Chain (HC) Sequences - optimizedLC (opt) 4910 nucleotide SEQ ID NO: 97GATATCGCACTGACCCAGCCAGCTTCAGTGAGCGGCTCACCAGGTCAGAGCATTACCATCTCGTGTACGGGTACTAGCAGCGATGTTGGTGGTTTTAATTATGTGTCTTGGTACCAGCAGCATCCCGGGAAGGCGCCGAAACTTATGATTCATGATGGTTCTAATCGTCCCTCAGGCGTGAGCAACCGTTTTAGCGGATCCAAAAGCGGCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCGGAAGACGAAGCGGATTATTATTGCCAGTCTTGGGATGTTTCTCCTATTACTGCTGTGTTTGGCGGCGGCACGAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAAAAGACAGTGGCCCCTACAGAATGTTCATAGLC4910 (BHQ880) polypeptide SEQ ID NO: 111DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSWDVSPITAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSLC (opt) 4945 nucleotide SEQ ID NO: 98GATATCGCACTGACCCAGCCAGCTTCAGTGAGCGGCTCACCAGGTCAGAGCATTACCATCTCGTGTACGGGTACTAGCAGCGATCTTGGTGGTTATAATTATGTGTCTTGGTACCAGCAGCATCCCGGGAAGGCGCCGAAACTTATGATTTATGATGTTAATAATCGTCCCTCAGGCGTGAGCAACCGTTTTAGCGGATCCAAAAGCGGCAACACCGCGAGCCTGACCATTAGCGGCCTGCAAGCGGAAGACGAAGCGGATTATTATTGCCAGACTTATGATCAGATTAAGTTGTCTGCTGTGTTTGGCGGCGGCACGAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAAAAGACAGTGGCCCCTACAGAATGTTCATAGLC4945 (BHQ892) polypeptide SEQ ID NO: 112DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTYDQIKLSAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLIPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSLC (opt) 4946 nucleotide SEQ ID NO: 99GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGAATCTTTTTTCTCCTTATCTGGCTTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGTGCTTCTAATCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTGTATTATTGCCAGCAGTATCTTACTCTTCCTCTTACCTTTGGCCAGGGTACGAAAGTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACACCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGLC4946 (BHQ898) polypeptide SEQ ID NO: 113DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYLTLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECLC (opt) 5145 nucleotide SEQ ID NO: 100GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGAATCTTTTTTCTCCTTATCTGGCTTGGTACCACCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGTGCTTCTAATCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTGTATTATTGCCAGCAGTATATGACTCTTCCTCTTACCTTTGGCCAGGGTACGAAAGTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGLC5145 (BHQ901) polypeptide SEQ ID NO: 114DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYMTLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHC (opt) 4910 nucleotide SEQ ID NO: 101CAGGCACAGGTCCAATTGGTGGAAACCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTTCTTATTGGATGTCTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATCTCTTATTCTGGTAGCAATACCCATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCCGAAGATACGGCCGTGTATTATTGCGCGCGTATGGGTATTGATCTTGATTATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC4910 (BHQ880) polypeptide SEQ ID NO: 115QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPCK HC (opt) 4945 nucleotide SEQ ID NO: 102CAGGCACAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTTCTTCTTATTGGATGTCTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGTTATCTCTTCTGATTCTAGCTCTACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTCATGGTATTGATTTTGATCATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC4945 (BHQ892) polypeptide SEQ ID NO: 116QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVISSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSKEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK HC (opt) 4946 =5145 nucleotide SEQ ID NO: 103CAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAACTACGGCATGACCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGGCATCAGCGGCAGCGGCAGCTACACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCCGGACCATCTACATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC4946 = 5145 (BHQ898/901) polypeptide SEQ ID NO: 117QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSKEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 13B Light Chain (LC) and Heavy Chain (HC) Sequences - parentalLC (parental) 4910 nucleotide SEQ ID NO: 104GATATCGCCCTGACCCAGCCCGCCAGCGTGTCCGGCAGCCCTGGCCAGAGCATCACCATCAGCTGTACCGGCACCAGCAGCGATGTGGGCGGCTTCAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCCACGACGGCAGCAATAGACCCAGCGGCGTGTCCAATAGATTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACCATCAGCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGCCAGAGCTGGGATGTGAGCCCCATCACCGCCGTGTTTGGCGGCGGAACAAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGACCACCGTGGAAAAGACAGTGGCCCCTACAGAATGTTCATAGLC (parental) 4945 SEQ ID NO: 105GATATCGCCCTGACCCAGCCCGCCAGCGTGTCCGGCAGCCCTGGCCAGAGCATCACCATCAGCTGTACCGGCACCAGCAGCGACCTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGAACAACAGACCTAGCGGCGTGTCCAACAGATTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACCATCTCTGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGCCAGACCTACGACCAGATCAAGCTGTCCGCCGTGTTTGGCGGCGGAACAAAGCTTACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACAACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAAAAGACAGTGGCCCCTACAGAATGTTCATAGLC (parental) 4946 nucleotide SEQ ID NO: 106GACATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGAACCTGTTCAGCCCTTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACGGCGCCAGCAACAGAGCCACCGGCGTGCCCGCCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGCCAGCAGTACCTGACCCTGCCCCTGACCTTCGGCCAGGGCACCAAGGTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGLC (parental) 5145 nucleotide SEQ ID NO: 107GACATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGAACCTGTTCAGCCCTTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACGGCGCCAGCAACAGAGCCACCGGCGTGCCCGCCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCTGAGGATTTCGCCGTGTACTACTGCCAGCAGTACATGACCCTGCCTCTGACCTTCGGCCAGGGCACCAAGGTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGHC (parental) 4910 nucleotide SEQ ID NO: 108CAGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGGCATCAGCTACAGCGGCAGCAATACCCACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCCGGATGGGCATCGACCTGGATTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC (parental) 4945 nucleotide SEQ ID NO: 109CAGGCCCAGGTGCAGCTGGTGGAGAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGTGATCAGCAGCGATAGCAGCAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGGCACGGCATCGACTTCGACCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACCGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA HC (parental) 4946 = 5145 nucleotide SEQ ID NO: 110CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAATAATTATGGTATGACTTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATCTCTGGTTCTGGTAGCTATACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTACTATTTATATGGATTATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA

Example 9 Bioactivity Assays

The biological activity of a neutralizing anti-DKK1/4 antibody ismeasured in a reporter gene assay, using the genetically modified cellline HEK293 T/17 STF_(—)70 IRES_Krm_(17) called SuperTopflash Krm17.This cell line is derived from the human embryonic kidney cell HEK293and is stably transfected with i) a reporter construct in which thepromoter TCF is fused upstream of the firefly luciferase gene and ii) aconstruct leading to overexpression of Krm on the surface of this cell.In this cell line, exposure to the Wnt protein stimulates the expressionof luciferase in a dose-dependent manner. Addition of graded amounts ofanti-DKK1/4 antibody to a fixed, sub-maximal dose of DKK1 in thepresence of Wnt causes an increase in the expression of luciferaseduring an incubation period of sixteen hours. At the end of this period,the amount of luciferase is quantified based on its enzymatic activityin the cell lysate. Luciferase catalyses the conversion of the substrateluciferin to oxyluciferin, a chemiluminescent product. The resultantglow-type chemi-luminescence is then determined with an appropriateluminometer.

The biological potency of a neutralizing anti-DKK1/4 antibody testsample is determined by comparing its ability to increase the luciferaseexpression to that of a reference standard. The samples and standard arenormalized on the basis of protein content. Relative potency is thencalculated using a parallel line assay according to the EuropeanPharmaco-poeia 1. The final result is expressed as relative potency (inpercent) of a sample compared to the reference standard.

Example 10 In Vitro Activity on Relevant Biological Targets

Lead FAbs are selected that have affinities in the low nanomolar rangeand potent activity in the cellular assay. The physiological bindingpartners for DKK1 are LRP5/6 (K_(a)-340 pM) and Kremen 1 and 2(K_(a)-280 pM) [Mao 2001][Mao 2002]. Given these high affinityinteractions, it is desirable to further improve affinity in order tobetter compete with the physiological DKK1 interactions. To increaseaffinity and biological activity of the selected FAbs, CDR-L3 and CDR-H2regions are optimized in parallel by cassette mutagenesis usingtrinucleotide directed mutagenesis [Virnekas 1994][Knappik 2000][Nagy2002].

Following affinity maturation, a FAb is selected that has low picomolaraffinity, reactivates DKK1 inhibited wnt signaling with an EC50 under 1nM, and cross reacts with cynomolgus monkey, mouse, and rat DKK1. Thevariable regions of this FAb are then engineered into two differenthuman IgG1 frameworks.

Anti-DKK1/4 antibody has high affinity for human DKK1 (2 pM) withbinding kinetics typical for an antibody of this affinity. See FIG. 6.

FIG. 6. METHODS: The binding affinity and kinetics of lead candidatesand rhDKK1 (recombinant human DKK1) (Batch BTP7757) are measured usingsurface plasmon resonance with a Biacore T100 (Biacore, Uppsala, Sweden)instrument containing a CM5 (S) sensor chip (Cat#BR-1006-68). Anti-HumanIgG1 Fc (Jackson Immuno Research, Cat#109-006-098) is immobilized ontoeach flow cell, followed by capture of a lead candidate at an expectedcapture of about 100 RU. Finally, six concentrations of DKK1 (range0.195-6.25 nM), with one repeat concentration, is run over the chip.Flow cells are activated for binding of DKK1 for 240 seconds, anddisassociation is followed for 30 minutes. The normalized data(background subtracted) are fit to a 1:1 binding with mass transportmodel using Kinetics analysis in BIA evaluation 1.0 software. Thisexperiment is carried out in triplicate, and data presented are theaverage of these three experiments with standard deviation.

Example 11 Epitope Mapping

Mature DKK1 is a 266 amino acid protein with two cysteine rich regions(Cys-1 and Cys-2). The Cys-2 domain is responsible for binding both LRPsand Kremen proteins and is necessary and sufficient for inhibition ofWnt signaling [Li 2002] [Brott 2002] Immunoprecipitation experiments(FIG. 7A, 7B) demonstrate that anti-DKK1/4 antibody binds specificallyto the Cys-2 domain, but not the Cys-1 domain. anti-DKK1/4 antibody isonly weakly active in Western blotting with denatured DKK1 and in apeptide mapping experiment is not found to specifically bind any of theoverlapping 15 amino acid peptides covering the length of the protein(JTP), suggesting that anti-DKK1/4 antibody likely recognizes anon-linear epitope within Cys-2.

FIG. 7(A) shows a schematic representation of full-length and truncatedDKK1. Full-length (FL, containing residues 1-266), carboxyl terminaltruncated (ΔC, containing residues 1-185), and amino terminal truncated(ΔN, containing residues 1-60 plus residues 157-266), are fused with anHA epitope at their C termini, and cloned into a mammalian expressionvector under the control of the cytomegalovirus (CMV) promoter. FIG.7(B) depicts binding of a neutralizing anti-DKK1/4 antibody and DKK1proteins. Conditioned medium from transiently transfected Hek293 cellsexpressing containing full length, amino truncated, carboxyl terminaltruncated DKK1 proteins are incubated with anti-lysozyme IgG1 control orthe anti-DKK1/4 antibodies for 2 hrs at room temperature, andimmunocomplexes are collected on protein G beads, resolved by SDS-PAGE,transferred, and blotted with an anti-HA antibody. 1/10 of total inputis loaded as control.

Example 11 Epitope Mapping—N-Glycosylation

A number of proteins within the Wnt signaling pathway are covalentlymodified by post-translational enzymes which regulate their cellularactivity. DKK family members, including

DKK1, are modified by N-glycosylation [Krupnik 1999]. DKK1 has onetheoretical N-linked glycosylation site at amino acid 256 within theCys-2 domain. Given the highly conserved nature of the Cys-2 domain, andthe potential binding site of both DKK1 for LRP6 and anti-DKK1/4antibody for DKK1 we sought to determine if anti-DKK1/4 antibodyrecognized the N-glycosylated form of DKK1. An ELISA demonstrates thatanti-DKK1/4 antibody recognizes the N-glycosylated form of rhDKK1 muchbetter then the specifically N-linked de-glycosylated form of rhDKK1TABLE 14A. While the same proteins are recognized equally well with asecond antibody (anti-HIS), directed towards the fused epitope tagregion of the recombinant protein. This difference in affinity isquantitated by using surface plasmon resonance and found anti-DKK1/4antibody to have 100 fold higher KD to the glycosylated rhDKK1, then tothe de-glycosylated protein, see TABLE 14B.

TABLE 14A Percent Binding—Glycosylation dependence of anti-DKK1/4antibody binding to DKK1 Antibody WT DKK1 DKK1 (dyglycosylated) anti-HIStag 100% 100% anti-DKK1/4 100% 12-18%

TABLE 14B surface plasmon resonance Protein Ka (1/Ms) Kd (1/s) KD (M) WTDKK1 7.449E+6 2.319E−5 3.113E−12 DKK1 (N-degly) 1.424E+6 3.071E−42.157E−10

The binding of anti-DKK1/4 antibody to wild type (WT) rhDKK1 (HEK HISepitope tagged Batch#BTP7757) and N-linked deglycosylated (N-DEGLY,N-linked deglycosylated with the enzyme PNGase F (Sigma, Cat#E-DEGLY)rhDKK1 is measured by ELISA. Briefly, a high binding ELISA (Nunc#442404)plate is coated with 1 μg/ml WT or N-DEGLY DKK1. The ratio of bothanti-DKK1/4 antibody and anti-HIS antibody binding to WT DKK1 ascompared to their respective binding of N-DEGLY is shown. Thisexperiment is carried out with three different concentrations (data forone representative concentration is shown), all concentrations hadsimilar results. B. The binding affinity and kinetics of anti-DKK1/4antibody to both WT and N-DEGLY DKK1(HEK293 Batch#BTP7757) are measuredusing a Biacore T100. anti-DKK1/4 antibody consistently had a 100 foldlower affinity for N-DEGLY DKK1 then it does for WT DKK1.

Example 12 Percent Identity of DKK Family Members

The human Dickkopf family consists of four paralogs (see Table 15),three of which (DKK1, 2, & 4) bind to LRP6 and Kremen proteins, induceinternalization of LRP5/6 and inhibit canonical Wnt signaling [Mao 2001][Mao 2003]. DKK2 also synergizes with LRP6 overexpression to enhance Wntsignaling, but co-expression of LRP6 and Kremen2 restores DKK2inhibition of the pathway [Mao 2003]. Thus DKK2 can act as both anagonist and an antagonist depending on the cellular context. DKK3 is theleast conserved of the family members, including within the Cys-2 domainresponsible for LRP5/6 and Kremen interactions and is distinct from theother DKK family members as it does not bind LRPs or Kremens and doesnot block Wnt signaling [Mao 2001] [Mao 2003].

TABLE 15 Percent identity of DKK family members across the whole proteinand within Cys-2 domains. DKK2 DKK3 DKK4 Whole Protein DKK1 38.7 15.532.5 DKK2 — 13.4 34.6 DKK3 — — 15.1 Cys-2 Domain DKK1 69.3 23.0 56.6DKK2 — 24.1 54.7 DKK3 — — 20.8

Homology among members of the DKK family is evaluated (Vector NTIAdvanced 9.1.0) using AlignX algorithm for pairwise sequence alignmentscomparing ratios of amino acid identities. Gap opening and gap extensionpenalties of 10 and 0.1 respectively are applied. This evaluationincluded comparisons of whole proteins, as well as comparisons of Cys-2domains only. As indicated in the table above, DKKs 1, 2 and 4 share30-40% amino acid sequence homology across the entire protein.Comparison of Cys-2 domains alone shows DKKs 1 and 2 share 69% homologywithin this region, while DKK4 shares roughly 57% with the same domainof DKKs 1 and 2. DKK3 shows the lowest level of homology to other familymembers. Amongst all members homology within the Cys-2 domain isgreatest.

Example 13 Affinity of Anti-DKK1/4 Antibody for Human DKK Family Members

In addition to binding DKK1, anti-DKK1/4 antibody also binds to DKK4,see Table 16. While the affinity for DKK4 is approximately 100 fold lessthan for DKK1, it is still subnanomolar and therefore likelybiologically and clinically relevant. Of note, neither DKK2 nor DKK4conserve the Asparagine residue that is predicted to be targeted forglycosylation in the Cys-2 domain of DKK1. Preliminaryimmunoprecipitation experiments suggest that anti-DKK1/4 antibody doesnot specifically bind DKK2. The binding affinity of anti-DKK1/4 antibodybinding to DKK2 will be determined following successful purification ofDKK2. Consistent with the distinct function and binding properties ofDKK3, anti-DKK1/4 antibody does not bind DKK3.

TABLE 16 Affinity of anti-DKK1/4 antibody for human DKK family membersDKK Family Member K_(D) DKK1 2.0 × 10-12M (±0.7) DKK2 ND DKK3 NSB DKK42.97 × 10-10M (±1.5)

The binding affinity and kinetics of anti-DKK1/4 antibody for othermembers of human DKK family of proteins are measured using a BiacoreT100. As before, experiments are carried out in triplicate for proteinswith significant binding to anti-DKK1/4 antibody and are reported as theaverage of three experiments with standard deviation. DKK3, which is theleast homologous family member, did not have binding that is detectableabove background levels and so is considered NSB (No Significantbinding). Likewise, recent data suggests that an anti-DKK1/4 antibody ofthe invention also has no significant binding to DKK2.

Example 14 Anti-DKK1/4 Antibody Blocks DKK1 Binding to LRP6

DKK1 mediates its Wnt antagonist activity through interactions withLRP5/6 and Kremen, inducing internalization and blocking Wnt inducedinteraction of LRP5/6 with Frizzled receptors. anti-DKK1/4 antibodycompetitively inhibits DKK1 binding to LRP6 in a competition ELISA assayin FIG. 8.

HEK293T cells do not express sufficient levels of endogenous LRP5 or 6to allow visualization of DKK1 binding. However, upon co-transfection ofLRP6 with a surface trafficking chaperone protein, MESD, GFP-tagged DKK1can be detected on the cell surface, illustrating the specific nature ofthe DKK1/LRP6 interaction. MOR04910, which shares the same variableregions as anti-DKK1/4 antibody, specifically blocks this interaction.

The ability of anti-DKK1/4 antibody to inhibit DKK1 binding directly toLRP6 is measured by ELISA. Briefly, non-treated plates (Fisher,Cat#12565501) are coated with 1 μg/ml of recombinant LRP6 (R&D SystemsCat#1505-LR), then 500 ng/ml of rhDKK1 and a concentration curve ofeither anti-DKK1/4 antibody or hIgG1 (anti-lysozyme MOR3207, ACE10915)are pre-incubated on ice for 30 minutes after which they are placed ontoLRP6 coated plates for 2 hours. Plates are washed and the level of DKK1binding is detected with anti-DKK1 antibody (R&D Systems AF1096). Shownare the raw OD values (background subtracted). Increasing concentrationsof anti-DKK1/4 antibody inhibits DKK1 binding directly to LRP6 in a dosedependent manner, while increasing concentrations of hIgG1 does notblock DKK1 binding to LRP6.

The ability of MOR04910 to inhibit DKK1/LRP6 binding on cell surface ismeasured by fluorescence microscopy. HEK293T cells are either mocktransfected and transiently transfected with plasmids encoding LRP6 andMESD. Cells are incubated with DKK1-GFP conditioned medium together withanti-lysozyme FAb or anti-DKK1 FAb MOR04910 for 1 hour at 37° C., andexamined by fluorescence microscopy. GFP fluorescence reflects DKK1-GFPbinding to overexpressed LRP6 on the plasma membrane. The anti-DKK1/4antibody blocks DKK1 interactions with LRP6 on cell surfaces.

Example 14 Reporter Assays—Reactivation of DKK1 Inhibited TCF/LEF GeneTranscription

Canonical Wnt signaling culminates in beta-catenin translocation to thenucleus where it associates with transcription factors of the TCF/LEFfamily resulting in enhanced transcription of Wnt-responsive genes. Areporter assay is established using a TCF/LEF responsive promoterdriving Luciferase gene transcription, facilitating detection of Wntpathway modulation. DKK1 effectively blocks luciferase activity inducedby Wnt3A conditioned media (CM) in this assay. Anti-DKK1/4 antibodyreactivates DKK1 suppressed Wnt signaling with an apparent EC50 of 0.16nM FIG. 9. Since the assay requires about 1 nM of DKK1 for completesuppression and the affinity of the antibody is 2 pM, it is likely thatthis EC50 reflects the sensitivity of the assay and relative amounts ofeach protein rather than an absolute limit of anti-DKK1/4 antibodycompetition.

293T cells stably transfected with SuperTopflash reporter and Kremen aretreated with 10 ng/ml of rhDKK1, 50% Wnt3a conditioned medium, andvarious amounts of anti-DKK1/4 antibody antibody. Eighteen hours later,luciferase activity is measured by the Bright-Glo luciferase assay kit(Promega).

Example 15 Reporter Assays—Reversal of DKK1 Inhibited AlkalinePhosphatase Secretion in Pre-Osteoblast-Like Cells

To determine whether anti-DKK1/4 antibody blocks DKK1 functions in amore physiological relevant setting, an in vitro assay is established tomeasure Wnt-mediated osteoblast differentiation of the pluripotent mousecell line C3H10T1/2 (10T1/2), see FIG. 10. Upon osteoblastdifferentiation the 10T1/2 cells secrete alkaline phosphatase (AP), aphenomena which can be inhibited by DKK1. Anti-DKK1/4 antibody, but notIgG control, blocks DKK1 suppression of 10T1/2 differentiation in thepresence of Wnt3A conditioned medium.

Wnt has been reported to induce proliferation and inhibit apoptosis in anumber of cell contexts and activation of the Wnt pathway, as indicatedby beta-catenin stabilization or nuclear localization, is frequentlyassociated with tumor progression. Furthermore, downregulation of DKK1in some cancers (e.g. colon carcinoma and melanoma) [Gonzalez-Sancho2005] [Kuphal 2006], has lead some investigators to suggest that DKK1may be a tumor suppressor for some cancers. To test whether DKK1 haseffect on tumor proliferation or survival, tumor cell lines are treatedwith anti-DKK1/4 antibody and analyzed for changes in growth. No tumorcell line tested is found to be significantly affected by addition ofanti-DKK1/4 antibody.

The effect of anti-DKK1/4 antibody on the survival and proliferation ofseveral cancer cell lines is assessed in vitro. In this assayanti-DKK1/4 antibody (100 μg/ml) is incubated with a tumor cell line,after three days cell number is assessed by quantitation of ATP(Promega, Cell Titer Glo Assay®), as a measure of metabolically activecells with a linear relationship to cell number. This assay is carriedout in three different serum concentrations (serum free, minimal growth,and complete growth). No significant changes, as compared to untreatedand hIgG1 treated cells are found. Cell line supernants are analyzed forDKK1 expression by ELISA.

Example 16 Species Crossreactivity and Neutralization of DKK1

A neutralizing anti-DKK1/4 antibody is selected not for its highaffinity against human DKK1 and neutralizing ability, but also basedupon its crossreactivity with other species that might be used forefficacy and safety studies. anti-DKK1/4 antibody crossreacts withmouse, rat, and cynomolgus monkey (cyno, Macaca fascicularis) DKK1 withsimilar affinity as for human DKK1, see Table 17. Moreover, anti-DKK1/4antibody neutralizes all four species' DKK1-mediated Wnt suppressiveactivity (Table 17), suggesting that these species should be relevantfor both safety and efficacy models.

TABLE 17 Species crossreactivity and neutralization of DKK1 Reactivationof wnt3a signaling (TOPFlash) DKK1 Protein K_(D) [pM] EC50 (pM) Human 1780.6 Cynomolgus 7 54.2 Mouse 10 60.5 Rat 16 255

Affinity determination for Human, Cynomolgus, Mouse, and Rat DKK1 isassayed by Solution Equilibrium Titration (SET) using the M-384 SERIES®analyzer (BioVeris, Europe). For KD determination by SolutionEquilibrium Titration (SET), monomer fractions (at least 90% monomercontent, analyzed by analytical SEC; Superdex75, Amersham Pharmacia) ofIgG protein are used. Electrochemiluminescence (ECL) based affinitydetermination in solution and data evaluation are basically performed asdescribed by [Haenel et al., 2005], the binding fit model is applied asmodified according to [Piehler et al., 1997]). A constant amount ofMOR4910 IgG is equilibrated with different concentrations (serial 3ndilutions) of human DKK1 (4 nM starting concentration) in solution.Biotinylated human DKK1 coupled to paramagnetic beads (M-280Streptavidin, Dynal) and BV-tag™ (BioVeris Europe, Witney, Oxfordshire,UK) labelled goat anti-human (Fab)′2 polyclonal antibody is added andincubated for 30 min. Subsequently, the concentration of unbound IgG isquantified via ECL detection using the M-384 SERIES® analyzer (BioVeris,Europe). Affinity determination to rat, mouse, and cynomolgus DKK1 isperformed essentially as described above using mouse, rat, andcynomolgus DKK1 as analyte in solution instead of human DKK1. Fordetection of free IgG molecules, biotinylated human DKK1 coupled toparamagnetic beads is used. MOR4910 and anti-DKK1/4 antibody neutralizehuman DKK1 (Novartis) with equivalent EC50, anti-DKK1/4 antibody alsoneutralizes monkey (Novartis), mouse(R&D Systems 1765-DK-010) and rat(Novartis) DKK1. The TOPFLASH reporter assay to human, rat, mouse, andcynomolgus DKK1 is performed essentially as described above (FIG. 9)using each species recombinant DKK1 as the inhibitor of Wnt conditionedmedia, instead of human DKK1. Rat recombinant DKK1 required higherconcentrations of protein to achieve significant inhibition of theTOPFLASH assay.

Example 17 Effect of Anti-DKK1 Antibody on Intratibial Growth ofPC3M2AC6 Xenografts

Prostate tumor metastases are unique among bone metastases in that theyare overwhelmingly osteoblastic rather than osteolytic [Keller 2001].However, even predominantly osteoblastic bone metastases have underlyingregions of osteolysis and frequently have low bone mass densities (BMD)especially when patients are on androgen ablation therapy [Saad 2006].Recently, it is demonstrated that DKK1 can act as a switch, wherebyexpression of DKK1 enhances osteolytic properties of a mixedosteoblastic/osteolytic prostate tumor cell line (C4-2B). In addition,shRNA suppression of DKK1 inhibited osteolytic activity of apredominantly osteolytic prostate tumor cell line (PC3) [Hall 2005][Hall 2006]. DKK1 knockdown also inhibited intratibial growth of thetumor xenograft, leading the authors to speculate that osteolyticactivity may be important for establishing a metastatic niche, butsubsequent loss of DKK1 in prostatic metastases converts the tumor to anosteoblastic phenotype.

An osteolytic prostate tumor model is adapted from a method by [Kim2003]. A variant of the osteolytic prostate tumor cell line (PC3M) thatstably expresses luciferase (PC3M2AC6) is injected into the tibia ofmice. The growth of the tumor is monitored by luciferase while changesin bone are monitored by micro-computerized tomography (micro-CT) andhistology. Rather than enhancing tumor growth, anti-DKK1/4 antibodytrended toward inhibition of tumor growth. While the inhibition is notsignificant in any one study, it has occurred consistently in 5/5studies conducted to date, a representative study showing effects of 3doses of anti-DKK1/4 antibody on tumor growth is shown FIG. 11. Asimilar non-significant trend toward inhibition occurred withanti-DKK1/4 antibody treated mice with subcutaneous PC3M2AC6 xenografts.

Treatments are started on day 5 post implantation (0.2 millioncells/animal). anti-DKK1/4 antibody is administered i.v., at doses of20, 60, and 200 μg/mouse/day, q.d., 3 times a week for 2 weeks. ControlIgG is administered i.v., at 200 μg/mouse/day, q.d., 3 times a week for2 weeks. Vehicle control (PBS) is administered i.v., q.d., 3 times aweek for 2 weeks. Final efficacy data and body weight change arecalculated after treatment.

Using this model, we found that an anti-DKK1/4 antibody inhibitstumor-induced cortical bone damage. Effects on trabecular bone areconfounded in this model by the observation that both tumor implants andsham implants cause mechanical damage to the bone that result in aninitial increase in woven bone which is later remodeled causing adecrease in apparent bone volume. Relative effects of newly formed wovenbone and trabeculae on overall bone volume/trabecular volume (BV/TV)ratios are therefore obscured. However, it is clear that anti-DKK1/4antibody increases the production of bone in both tumor and shamimplanted tibias and inhibits or delays the decrease in bone volumeaccompanying remodeling. Using the same tumor-induced osteolytic model,anti-DKK1/4 antibody demonstrates equivalent anti-osteolytic activity asZometa, see FIG. 12. The bone metabolic effects of anti-DKK1/4 antibodyare dose responsive in the range from 20-200 μg/mouse, with a minimallyefficacious dose between 20 and 60 μg/mouse, see FIG. 13. Together thesedata suggest that anti-DKK1/4 antibody should have an impact intumor-induced osteolytic disease, but may also be effective in non-tumorbone diseases such as osteoporosis or enhancing repair of bonefractures.

Example 18 Anti-DKK1/4 Antibody Maintains Elevated Bone Density in BothTumor and Sham Implanted Tibias

In an effort to assess pharmacodynamic markers of efficacy in the micethree serum markers of bone metabolism are analyzed: osteocalcin (OC),osteoprotegerin (OPG), and secreted receptor activator of nuclear factorKB ligand (sRANKL). These osteoblast markers are used rather than themore typical osteoclast markers due to the expected mechanism of actionof the antibody. However, no consistent changes are detected in animalswith tumor versus naïve animals. No correlation of bone loss, asmeasured by micro-CT or IHC, with any of these markers are consistentlyobserved.

Representative examples of MicroCT reconstructions of the tibias oftreated mice are shown in FIG. 12A. Cortical damage is scored from 0=nodamage to 3=severe damage. FIG. 12B. Cortical damage in tumor-implantedtibias is manually scored by microCT analysis that are blinded withrespect to the study groups. No cortical damage is observed in any ofthe sham implanted legs.

Methods: Female nude mice at age of 12 weeks old are implantedintatibially with 2×10⁵ PC-3M2AC6 cells in the left tibia andsham-injection in the right tibia. Treatments started on day 5 postimplantation. NVP-anti-DKK1/4 antibody-NX (anti-DKK1/4 antibody) and IgGcontrol are administered i.v., at doses of 200 μg/mouse/day, q.d., 3times a week for 2 weeks. Vehicle (PBS) control is also administeredi.v., q.d., 3 times a week for 2 weeks. Animals are scanned at day 7,14, and 18 post tumor implantation using the μ-CT VivaCT40 Scanner(SCANCO, Switzerland). Trabecular bone density (BV/TV) is analyzed asdescribed in methods. An asterisks (*) indicates statistical significantdifference from both vehicle and IgG controls (n=12) at the same timepoint at p<0.05.

In FIG. 13, to determine the bone mass, the secondary spongiosa of thetibia is imaged with the Zeiss Imager Z.1 and Axiovision software basedon Giemsa stain. The readout is based on the percent calcified bone inthe entire field. Every column represents the mean and standarddeviation of the stated number of animals. In the PBS, IgG, andanti-DKK1/4 antibody treated groups, only animals with tumor areanalyzed. Right legs did not have sham injections and left legs hadtumor. Statistic: Dunnett Multiple Comparisons Test One-Way ANOVA. Leftlegs or right legs compared to the respective leg in the PBS groupp<0.05*,p<0.01**,p>0.05 n.s.

FIG. 14 shown an anti-DKK1/4 antibody's anabolic bone efficacy is dosedependent with minimal efficacious dose between 20 and 60 μg/mouse3x/week. Female nude mice at age of 12 weeks old are implantedintratibially with 2×105 PC-3M2AC6 cells in the left tibia andsham-injection in the right tibia. Treatments started on day 6 postimplantation. NVP-anti-DKK1/4 antibody-NX (anti-DKK1/4 antibody) isadministered i.v., at doses of 20, 60, and 200 μg/mouse/day, q.d., 3times a week for 2 weeks. Control IgG is administered i.v., at 200μg/mouse/day, q.d., 3 times a week for 2 weeks. Vehicle control (PBS) isadministered i.v., q.d., 3 times a week for 2 weeks. Animals are scannedat day 7 and 20 post tumor implantation using the μ-CT VivaCT40 Scanner(SCANCO, Switzerland). Trabecular bone density (BV/TV) is analyzed asdescribed in methods. * indicates statistical significant differencefrom all controls including, vehicle, IgG, drill only, and naïve animalsat the same time point at p <0.05.

Example 18 Biomarker Status

DKK1 Biomarkers

The RNA expression pattern of DKK1 has been described. Krupnik (1999)showed expression in placenta by Northern Blot analysis, with noexpression detected in heart, brain, lung, liver, skeletal muscle orpancreas. Wirths (2003) showed lack of RNA expression in liver, kidney,and breast, although RNA expression is seen in a subset ofhepatoblastomas and Wilms' Tumors. Workers examining gastrointestinaltract expression of DKK1 by RNA in situ hybridization showed noexpression in stomach and colon, whether normal or malignant (Byun2006).

RNA expression analysis in mice revealed high DKK1 expression levels inbone, medium expression in fetus and placenta, and weak expression inbrown adipose tissue, thymus and duodenum [Li 2006].

DKK1 protein expression is evaluated in myeloma specimens using the samegoat antibody employed in the current study (Tian, 2003). In this paper,expression is seen in myeloma cells of patients with low grademorphology; DKK1 protein expression is not detected in the bone marrowbiopsy specimens of five control subjects.

Tissue distribution and species crossreactivity of the therapeuticantibody anti-DKK1/4 antibody is studied by screening it against aseries of normal human and monkey tissues. Both whole tissue sectionsand tissue microarrays are evaluated. Positive controls included acommercial antibody for DKK1 that is evaluated in the same tissue set.

DKK1_(—)15 (FITC conjugated anti-DKK1/4 antibody) and DKK1_(—)8 (FITCconjugated Goat anti-DKK1, R&D Systems, # AF1096, lots GBL013101 andGBL14111).

Other Biomarkers

Since little is known about the pathophysiologic role of DKK1, asignificant amount of effort is and has been focused on building up theknowledge base about the in vivo effects of anti-DKK1/4 antibody bybiomarker studies and how this could be exploited to further thedevelopment. Key areas of focus have included

1) Understanding the effect of anti-DKK1/4 antibody in normal andmetastatic bone metabolism by the measurement of circulating markers ofosteoclastic and osteoblastic activity.

2) Comparative expression levels of DKK1 in multiple myeloma and othertumors to confirm and expand target indications.

3) Effects at a gene expression level in key tissues like colon, bonemarrow, lung, skin and breast to assess beta-catenin activation.

Preliminary molecular epidemiology studies have confirmed increased DKK1serum levels in patients with multiple myeloma and support a POC in thisindication.

Based on existing knowledge, Table 18 provides the proposed potentialBiomarkers for an anti-DKK1/4 antibody.

TABLE 18 Biomarkers for DKK1 and DKK4 targets Surrogate Categories TumorBlood Tissue Pharmacodynamic N/A Free and anti-DKK1/4 Ab bound Adipose/(PD) DKK-1 levels skin Target Activation of beta-catenin Downstream NTx,CTx, PINP, Osteocalcin, Mechanism of Action RANKL, OPG, PTH, Vitamin D3,calcitonin Efficacy Serum M protein, NTx, CTx, PINP, Osteocalcin, Urinetotal M protein, RANKL, OPG, PTH, b2 microglobin, LDHcalcitoninBone-ALP, CICP, CTx, NTx, etc Predictive Markers DKK1expression DKK-1, CTx, PINP, Osteocalcin, Stratification RANKL, OPG,PTH, Vit D3, Preselection calcitonin DKK1 serum levels SafetyImmunogenicity Pharmacokinetic anti-DKK1/4 Ab

Example 19 Amino Acid Sequences of Heavy and Light Chain VariableRegions of Anti-DKK1 Antibodies

The amino acid sequences of the variable regions of the light and heavychains of anti-DKK1 antibodies, whose CDR regions are shown in Tables 5,6, 11A and 11B, are provided in full in Table 19.

TABLE 19Amino Acid Sequences of Heavy and Light Chain Variable Regions ofanti-DKK1 Antibodies (SEQ ID NOS: 2-39) MOR04454 VH: (SEQ ID NO: 2)QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGLHWVRQAPGKGLEWVSSISYYGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGSHMDKPPGYVFAFWGQGTLVTVSSMOR04454 VL: (SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQGIKNYLNWYQQKPGKAPKLLIGAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYYGMPPTFGQGTKVEIKRT MOR04455 VH: (SEQ ID NO: 3)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHYMDHWGQGTLVTVSS MOR04455 VL: (SEQ ID NO: 22)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLHWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYDSIPMTFGQGTKVEIKRT MOR04456 VH: (SEQ ID NO: 4)QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWGQGTLVTVSS MOR04456 VL: (SEQ ID NO: 23)DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQQYGDEPITFGQGTKVEIKRT MOR04461 VH: (SEQ ID NO: 5)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYWGQGTLVTVSS MOR04461 VL: (SEQ ID NO: 24)DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSTWDMTVDFVFGGGTKLTVLGQ MOR04470 VH: (SEQ ID NO: 6)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVISSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSS MOR04470 VL: (SEQ ID NO: 25)DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYASGNTKVVFGGGTKLTVLGQ MOR04516 VH: (SEQ ID NO: 7)QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIGWVRQMPGKGLEWMGIIYPTDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGIPFRMRGFDYWGQGTLVTVSSMOR04516 VL: (SEQ ID NO: 26)DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSSFVNWYQQLPGTAPKLLIGNNSNRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCASFDMGSPNVVFGGGTKLTVLGQ MOR04907 VH: (SEQ ID NO: 8)QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWGQGTLVTVSS MOR04907 VL: (SEQ ID NO: 27)DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYLSLPTTFGQGTKVEIKRT MOR04913 VH: (SEQ ID NO: 9)QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWGQGTLVTVSS MOR04913 VL: (SEQ ID NO: 28)DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYMTLPLTFGQGTKVEINRT MOR04946 VH: (SEQ ID NO: 10)QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWGQGTLVTVSS MOR04946 VL: (SEQ ID NO: 29)DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYLTLPLTFGQGTKVEIKRT MOR04910 VH: (SEQ ID NO: 11)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYWGQGTLVTVSS MOR04910 VL: (SEQ ID NO: 30)DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSWDVSPITAVFGGGTKLTVLGQ MOR04921 VH: (SEQ ID NO: 12)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYWGQGTLVTVSS MOR04921 VL: (SEQ ID NO: 31)DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTWATSPLSSVFGGGTKLTVLGQ MOR04948 VH: (SEQ ID NO: 13)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYWGQGTLVTVSS MOR04948 VL: (SEQ ID NO: 32)DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTWDSLSFFVFGGGTKLTVLGQ MOR04914 VH: (SEQ ID NO: 14)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVISSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSS MOR04914 VL: (SEQ ID NO: 33)DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYTYTPISPVFGGGTKLTVLGQ MOR04920 VH: (SEQ ID NO: 15)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSSIEHKDAGYTTWYAAGVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSSMOR04920 VL: (SEQ ID NO: 34)DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYASGNTKVVFGGGTKLTVLGQ MOR04945 VH: (SEQ ID NO: 16)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVISSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSS MOR04945 VL: (SEQ ID NO: 35)DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQTYDQIKLSAVFGGGTKLTVLGQ MOR04952 VH: (SEQ ID NO: 17)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVISSDSSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSS MOR04952 VL: (SEQ ID NO: 36)DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYDSPTDSVVFGGGTKLTVLGQ MOR04954 VH: (SEQ ID NO: 18)QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSVIEHKDKGGTTYYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGIDFDHWGQGTLVTVSSMOR04954 VL: (SEQ ID NO: 37)DIALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSYASGNTKVVFGGGTKLTVLGQ MOR04947 VH: (SEQ ID NO: 19)QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIGWVRQMPGKGLEWMGIIVPGTSYTIYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGIPFRMRGFDYWGQGTLVTVSSMOR04947 VL: (SEQ ID NO: 38)DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSSFVNWYQQLPGTAPKLLIGNNSNRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCASFDMGSPNVVFGGGTKLTVLGQ MOR05145 VH: (SEQ ID NO: 20)QVQLVESGGGLVQPGGSLRLSCAASGFTFNNYGMTWVRQAPGKGLEWVSGISGSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYMDYWGQGTLVTVSS MOR05145 VL: (SEQ ID NO: 39)DIVLTQSPATLSLSPGERATLSCRASQNLFSPYLAWYQQKPGQAPRLLIYGASNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYMTLPLTFGQGTKVEIKRT

The CDR and FR sections of the variable regions in Table 19 are alignedin Table 20A for heavy chains (SEQ ID NOS:2-20; VH3 is SEQ ID NO:125,VH5 is SEQ ID NO:126), Table 20B for kappa light chains (SEQ ID NOS: 21,22, 23, 27, 28 and 29; VK1 is SEQ ID NO:127 and VK3 is SEQ ID NO:128),and in Table 20C for lambda light chains (SEQ ID NOS: 24, 25, 30, 31,32, 33, 34, 35, 36 and 37; VL2 is SEQ ID NO:129 and VL1 is SEQ IDNO:130).

TABLE 20AAlignment of the Amino Acid Sequences of Heavy Chain Variable Regions of anti-DKK1 Antibodies (SEQ ID NOs: 2-20, 125-126)

TABLE 20BAlignment of the Amino Acid Sequences of Kappa Light Chain Variable Regions of anti-DKK1 Antibodies (SEQ ID NOs: 24-25, 30-37, 129-130)

TABLE 20CAlignment of the Amino Acid Sequences of Lambda Light Chain Variable Regions of anti-DKK1 Antibodies  (SEQ ID NOs:)

Consensus CDR sequences are provided at least in SEQ ID NOS:40-48 and inTable 11B. Additional consensus CDR sequences may be determined by oneskilled in the art from the alignments in Tables 20A-20C using standardmethods and methods provided herein.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that novel antibodies andimmunological fragments thereof have been described. Although particularembodiments have been disclosed herein in detail, this has been done byway of example for purposes of illustration only. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention.

What is claimed is:
 1. A composition comprising: (1) an isolatedantibody or a functional fragment thereof comprising: (a) CDR sequencesof a variable heavy chain comprising: CDR1 with a sequence comprisingamino acids 22 to 35 of SEQ ID NO: 11, CDR2 with a sequence comprisingamino acids 47 to 66 of SEQ ID NO: 11, and CDR3 with a sequencecomprising amino acids 99 to 105 of SEQ ID NO: 11, and (b) CDR sequencesof a variable light chain comprising: CDR1 with a sequence comprisingamino acids 23 to 36 of SEQ ID NO: 30, CDR2 with a sequence comprisingamino acids 48 to 58 of SEQ ID NO: 30, and CDR3 with a sequencecomprising amino acids 91 to 100 of SEQ ID NO: 30, wherein the antibodyor functional fragment thereof specifically binds to human DKK1 and/orhuman DKK4; and (2) a bisphosphonate.
 2. The composition according toclaim 1, wherein binding to DKK1 or DKK4 is determined in at least oneassay selected from: antagonism of Wnt-signaled transcription;electrochemiluminescence-based binding analysis; enzyme-linkedimmunosorbent assay binding; FMAT; SET; SPR; osteocalcin (OCN) serumconcentration; osteoprotegrin (OPG) serum concentration; procollagentype 1 nitrogenous propeptide (P1NP) serum concentration, ALPproduction; TopFlash or ameliorating osteolysis.
 3. The compositionaccording to claim 1, wherein the isolated antibody or functionalfragment is an isolated antibody.
 4. The composition according to claim1, which comprises a scaffold selected from an IgM and an IgG, whereinthe IgG is selected from an IgG1, an IgG2, and IgG3 or an IgG4.
 5. Thecomposition according to claim 4, wherein the IgM or the IgG is selectedfrom a polyclonal or monoclonal.
 6. The composition according to claim1, which is selected from a whole immunoglobulin or Fab fragment or scFvantibody fragment thereof, a heavy chain antibody, and anantigen-binding region thereof on a non-immunoglobulin scaffold.
 7. Thecomposition according to claim 1, wherein the bisphosphonate iszoledronic acid.
 8. A composition comprising: (1) an isolated antibodyor a functional fragment thereof comprising: (a) CDR sequences of avariable heavy chain comprising: CDR1 with a sequence comprising aminoacids 22 to 35 of SEQ ID NO: 11, CDR2 with a sequence comprising aminoacids 47 to 66 of SEQ ID NO: 11, and CDR3 with a sequence comprisingamino acids 99 to 105 of SEQ ID NO: 11, and (b) CDR sequences of avariable light chain comprising: CDR1 with a sequence comprising aminoacids 23 to 36 of SEQ ID NO: 30, CDR2 with a sequence comprising aminoacids 48 to 58 of SEQ ID NO: 30, and CDR3 with a sequence comprisingamino acids 91 to 100 of SEQ ID NO: 30, wherein the antibody orfunctional fragment thereof specifically binds to human DKK1 and/orhuman DKK4; (2) a bisphosphonate; and (3) a pharmaceutically acceptablecarrier or excipient.
 9. The composition according to claim 8, whereinthe isolated antibody or functional fragment is an isolated antibody.10. The composition according to claim 8, which comprises a scaffoldselected from an IgM and an IgG, wherein the IgG is selected from anIgG1, an IgG2, and IgG3 or an IgG4.
 11. The composition according toclaim 10, wherein the IgM or the IgG is selected from a polyclonal ormonoclonal.
 12. The composition according to claim 8, which is selectedfrom a whole immunoglobulin or Fab fragment or scFv antibody fragmentthereof, a heavy chain antibody, and an antigen-binding region thereofon a non-immunoglobulin scaffold.
 13. The composition according to claim8, wherein the bisphosphonate is zoledronic acid.
 14. A compositioncomprising: (1) an immunoconjugate comprising a first component which isan antibody or functional fragment thereof comprising: (a) CDR sequencesof a variable heavy chain comprising: CDR1 with a sequence comprisingamino acids 22 to 35 of SEQ ID NO: 11, CDR2 with a sequence comprisingamino acids 47 to 66 of SEQ ID NO: 11, and CDR3 with a sequencecomprising amino acids 99 to 105 of SEQ ID NO: 11, and (b) CDR sequencesof a variable light chain comprising: CDR1 with a sequence comprisingamino acids 23 to 36 of SEQ ID NO: 30, CDR2 with a sequence comprisingamino acids 48 to 58 of SEQ ID NO: 30, and CDR3 with a sequencecomprising amino acids 91 to 100 of SEQ ID NO: 30, wherein the antibodyor functional fragment thereof specifically binds to human DKK1 and/orhuman DKK4; and (2) a bisphosphonate.
 15. The composition according toclaim 14, wherein the isolated antibody or functional fragment is anisolated antibody.
 16. The composition according to claim 14, whichcomprises a scaffold selected from an IgM and an IgG, wherein the IgG isselected from an IgG1, an IgG2, and IgG3 or an IgG4.
 17. The compositionaccording to claim 16, wherein the IgM or the IgG is selected from apolyclonal or monoclonal.
 18. The composition according to claim 14,which is selected from a whole immunoglobulin or Fab fragment or scFvantibody fragment thereof, a heavy chain antibody, and anantigen-binding region thereof on a non-immunoglobulin scaffold.
 19. Thecomposition according to claim 14, wherein the bisphosphonate iszoledronic acid.